Artificial chromosomes, uses thereof and methods for preparing artificial chromosomes

ABSTRACT

Methods for preparing cells that contain artificial chromosomes, methods for preparation of artificial chromosomes, methods for purification of artificial chromosomes, methods for targeted insertion of heterologous DNA into artificial chromosomes, and methods for delivery of the chromosomes to selected cells and tissues are provided. Also provided methods for producing transgenic plants and animals using the artificial chromosomes and the resulting transgenic organisms are provided.

RELATED APPLICATIONS

[0001] This application is a continuation of allowed U.S. applicationSer. No. 09/096,648, filed Jun. 12, 1998, which is a continuation ofU.S. application Ser. No. 08/629,822, filed Apr. 10, 1996. Accordinglybenefit of priority of both applications is claimed.

[0002] This application is related to U.S. application Ser. No.08/759,558, now U.S. Pat. No. 5,288,625 and to U.S. application Ser. No.08/375,271, filed Jan. 19, 1995, which is a continuation of U.S.application Ser. No. 08/080,097, filed Jun. 23, 1993 which is acontinuation of U.S. application Ser. No. 07/892,487, filed Jun. 3,1992, which is a continuation of U.S. application Ser. No. 07/521,073,filed May 9, 1990.

[0003] The subject matter of each of the above-listed U.S. applicationsis incorporated in its entirety by reference thereto.

FIELD OF THE INVENTION

[0004] The present invention relates to methods for preparing cell linesthat contain artificial chromosomes, to methods for isolation of theartificial chromosomes, targeted insertion of heterologous DNA into thechromosomes, isolation of the chromosomes, and delivery of thechromosomes to selected cells and tissues. Also provided are cell linesfor use in the methods, and cell lines and chromosomes produced by themethods.

BACKGROUND OF THE INVENTION

[0005] Several viral vectors, non-viral, and physical delivery systemsfor gene therapy have been developed (see, e.g., Mitani et al. (1993)Trends Biotech. 11:162-166). The presently available systems, however,have numerous limitations, particularly where persistent, stable, orcontrolled gene expression is required. These limitations include: (1)size limitations because there is a limit, generally on order of aboutten kilobases (kB), at most, to the size of the DNA insert (gene) thatcan be accepted by viral vectors, whereas a number of mammalian genes ofpossible therapeutic importance are well above this limit, especially ifall control elements are included; (2) the inability to specificallytarget integration so that random integration is required which carriesa risk of disrupting vital genes or cancer suppressor genes; (3) theexpression of randomly integrated therapeutic genes may be affected bythe functional compartmentalization in the nucleus and are affected bychromatin-based position effects; (4) the copy number and consequentlythe expression of a given gene to be integrated into the genome cannotbe controlled. Thus, improvements in gene delivery and stable expressionsystems are needed (see, e.g., Mulligan (1993) Science 260:926-932).

[0006] In addition, safe and effective gene therapy methods and vectorsshould have numerous features that are not assured by the presentlyavailable systems. For example, a safe vector should not contain DNAelements that can promote unwanted changes by recombination or mutationin the host genetic material, should not have the potential to initiatedeleterious effects in cells, tissues, or organisms carrying the vector,and should not interfere with genomic functions. In addition, it wouldbe advantageous for the vector to be non-integrative, or designed forsite-specific integration. Also, the copy number of therapeutic gene(s)carried by the vector should be controlled and stable, the vector shouldsecure the independent and controlled function of the introducedgene(s); and the vector should accept large (up to Mb size) inserts andensure the functional stability of the insert.

[0007] The limitations of existing gene delivery technologies, however,argue for the development of alternative vector systems suitable fortransferring large (up to Mb size or larger) genes and gene complexestogether with regulatory elements that will provide a safe, controlled,and persistent expression of the therapeutic genetic material.

[0008] At the present time, none of the available vectors fulfill theserequirements. Some of these characteristics, however, are possessed bychromosomes. Thus, an artificial chromosome would be an ideal vector forgene therapy, as well as for production of gene products that requirecoordination of expression of numerous genes or that are encoded bylarge genes, and other uses. Artificial chromosomes for expression ofheterologous genes in yeast are available, but construction of amammalian artificial chromosome has not been achieved. Such constructionhas been hindered by the lack of an isolated, functional, mammaliancentromere and uncertainty regarding the requisites for its productionand stable replication. Unlike in yeast, there are no selectable genesin close proximity to a mammalian centromere, and the presence of longruns of highly repetitive pericentric heterochromatic DNA makes theisolation of a mammalian centromere using presently available methods,such as chromosome walking, virtually impossible. Other strategies arerequired for production of mammalian artificial chromosomes, and somehave been developed. For example, U.S. Pat. No. 5,288,625 provides acell line that contains an artificial chromosome, a minichromosome, thatis about 20 to 30 megabases. Methods provided for isolation of thesechromosomes, however, provide preparations of only about 10-20% purity.Thus, development of alternative artificial chromosomes and perfectionof isolation methods as well as development of more versatilechromosomes and further characterization of the minichromosomes isrequired to realize the potential of this technology.

[0009] Therefore, it is an object herein to provide mammalian artificialchromosomes and methods for introduction of foreign DNA into suchchromosomes. It is also an object herein to provide methods forintroduction of the artificial mammalian chromosome into selected cells,and to provide the resulting cells, as well as transgenic animals andplants that contain the artificial chromosomes. It is also an objectherein to provide methods for gene therapy and expression of geneproducts using artificial chromosomes. It is a further object herein toprovide methods for constructing species-specific artificialchromosomes.

SUMMARY OF THE INVENTION

[0010] Mammalian artificial chromosomes (MACs) are provided. Alsoprovided are artificial chromosomes for other higher eukaryotic species,such as insects and fish, produced using the MACS are provided herein.Methods for generating and isolating such chromosomes. Methods using theMACs to construct artificial chromosomes from other species, such asinsect and fish species are also provided. The artificial chromosomesare fully functional stable chromosomes. Two types of artificialchromosomes are provided. One type, herein referred to as SATACs(satellite artificial chromosomes) are stable heterochromaticchromosomes, and the another type are minichromosomes based onamplification of euchromatin.

[0011] Artificial chromosomes permit targeted integration of megabasepair size DNA fragments that contain single or multiple genes. Thusmethods using the MACs to introduce the genes into cells, animals andtissues are also provided. The artificial chromosomes with integratedheterologous DNA are to be used in methods of gene therapy, in methodsof production of gene products, particularly products that requireexpression of multigene biosynthetic pathways, and also are intended fordelivery into the nuclei of germline cells, such as embryo-derived stemcells (ES cells) for production of transgenic animals.

[0012] Mammalian artificial chromosomes provide extra-genomic specificintegration sites for introduction of genes encoding proteins ofinterest and permit megabase size DNA integration so that, for example,genes encoding an entire metabolic pathway or a very large gene, such asthe cystic fibrosis (CF; ˜600 kb) gene, several genes, such as a seriesof antigens for preparation of a multivalent vaccine, can be stablyintroduced into a cell. Vectors for targeted introduction of such genes,including the tumor suppressor genes, such as p53, the cystic fibrosistransmembrane regulator gene (CFTR), anti-HIV ribozymes, such as ananti-HIV gag ribozyme, into the artificial chromosomes also provided.

[0013] The chromosomes provided herein are generated by introducingheterologous DNA that includes DNA encoding a selectable marker intocells, preferably a stable cell line, growing the cells under selectiveconditions, and identifying from among the resulting clones those thatinclude chromosomes with more than one centromere or that havechromosomes that are fragments of chromosomes that had more than onecentromere. The amplification that produces the additional centromereoccurs in cells that contain chromosomes in which the heterologous DNAhas integrated near the centromere in the pericentric region of thechromosome. The selected clonal cells are then used to generateartificial chromosomes.

[0014] In preferred embodiments, the DNA with the selectable marker thatis introduced includes sequences that target it to the pericentricregion of the chromosome. For example, vectors, such as pTEMPUD, whichincludes such DNA specific for mouse satellite DNA, are provided. Alsoprovided are derivatives of pTEMPUD that specifically target humansatellite sequences. Upon integration, these vectors can induce theamplification.

[0015] Artificial chromosomes are generated by culturing the cells withthe dicentric chromosomes under conditions whereby the chromosome breaksto form a minichromosome and formerly dicentric chromosome. Theartificial chromosomes (the SATACs) are generated, not from theminichromosome fragment as, for example, in U.S. Pat. No. 5,288,625, butfrom the fragment of the formerly dicentric chromosome.

[0016] Among the MACs provided herein are the SATACs, which areprimarily made up of repeating units of short satellite DNA and arefully heterochromatic, so that absent insertion of heterologous orforeign DNA, the chromosomes do not contain genetic information. Theycan thus be used as “safe” vectors for delivery of DNA to mammalianhosts because they do not contain any potentially harmful genes.

[0017] In addition to MACs methods for generating euchromaticminichromsomes and the use thereof are also provided herein. Methods forgenerating one type of MAC the minichromosome, previously described inU.S. patent No. U.S. Pat. No. 5,288,625, and the use thereof forexpression of heterologous DNA are provided. Cell lines containing theminichromosome and the use thereof for cell fusion are also provided.

[0018] In one embodiment, a cell line containing the mammalianminichromosome is used as recipient cells for donor DNA encoding aselected gene or multiple genes. The donor DNA is linked to a secondselectable marker and is targeted to and integrated into theminichromosome. The resulting chromosome is transferred by cell fusioninto an appropriate recipient cell line, such as a Chinese hamster cellline (CHO). After large scale production of the cells carrying theengineered chromosome, the chromosome is isolated. In particular,metaphase chromosomes are obtained, such as by addition of colchicine,and they are purified from the cell lysate. These chromosomes are usedfor cloning, sequencing and for delivery of heterologous DNA into cells.

[0019] Also provided are SATACs of various sizes that are formed byrepeated culturing under selective conditions and subcloning of cellsthat contain chromosomes produced from the formerly dicentricchromosomes. These chromosomes are based on repeating units 7.5 to 10 Mbreferred to herein as megareplicons, that are tandem blocks of satelliteDNA flanked by heterologous non-satellite DNA. Amplification produces atandem array of identical chromosome segments (each called an amplicon)that contain two inverted megareplicons bordered by heterologous(“foreign”) DNA. Repeated cell fusion, growth on selective medium and/orBrdU (5-bromodeoxyuridine) treatment or other genome destabilizingreagent or agent, such as ionizing radiation, including X-rays, andsubcloning results in cell lines that carry stable heterochromatic orpartially heterochromatic chromosomes, including a 150-200 Mb “sausage”chromosome, a 500-1000 Mb gigachromosome, a stable 250-400 Mbmegachromosome and various smaller stable chromosomes derived therefrom.These chromosomes are based on these repeating units and can includeheterologous DNA that is expressed.

[0020] Thus methods for producing MACs of both types are provided. Thesemethods are applicable to any higher eukaryotic cell, including mammals,insects and plants.

[0021] The resulting chromosomes can be purified by methods providedherein to provide vectors for introduction of the heterologous DNA intoselected cells for production of the gene product encoded by theheterologous DNA, for production of transgenic animals and plants or forgene therapy. Vectors for chromosome fragmentation are provided. Thesevectors will be used to produce an artificial chromosome that contains amegareplicon, a centromere and two telomeres and will be between about10 Mb and about 60 Mb, preferably between about 10 Mb-15 Mb and 30 Mb.Such artificial chromosomes may be produced by other methods. Isolationof the 7.5 Mb (or 15 Mb amplicon containing two 7.5 Mb inverted repeats)or a 30 Mb multimer thereof should provide a stable chromosomal vectorthat can be manipulated in vitro.

[0022] In addition, methods and vectors for fragmenting theminichromosomes and SATACs are provided. Such methods and vectors can beused for in vivo generation of smaller stable artificial chromosomes.Methods for reducing the size of the MACs to generate smaller stableself-replicating artificial chromosomes are also provided.

[0023] Methods and vectors for targeting heterologous DNA into theartificial chromosomes are also provided as are methods and vectors forfragmenting the chromosomes to produce smaller but stable andself-replicating artificial chromosomes. Vectors for targetedintroduction of heterologous DNA into artificial chromosomes areprovided.

[0024] The chromosomes are introduced into cells to produce stabletransformed cell lines or cells, depending upon the source of the cells.Introduction is effected by any suitable method including, but notlimited to electroporation, direct uptake, such as by calcium phosphate(see, e.g., Wigler et al. (1979) Proc. Natl. Acad. Sci. U.S.A.76:1373-1376; and Current Protocols in Molecular Biology, Vol. 1, WileyInter-Science, Supplement 14, Unit 9.1.1-9.1.9 (1990)). precipitation,uptake of isolated chromosomes by lipofection, by microcell fusion (see,EXAMPLES, see, also Lambert (1991) Proc. Natl. Acad. Sci. U.S.A.88:5907-5911, U.S. Pat. No. 5,396,767) or other suitable method. Theresulting cells can be used for production of proteins in the cells. Thechromosomes can be isolated and used for gene delivery.

[0025] Methods for isolation of the chromosomes based on the DNA contentof the chromosomes, which differs from the authentic chromosomes areprovided.

[0026] These artificial chromosomes can be used in gene therapy, geneproduct production systems, production of humanized organs, productionof transgenic plants and animals, including invertebrates, vertebrate,reptiles and insects, any organism or device that would employchromosomal elements as information storage vehicles, and also foranalysis and study of centromere function, for the production ofartificial chromosome vectors that can be constructed in vitro, and forthe preparation of species-specific artificial chromosomes. Theartificial chromosomes can be introduced into cells usingmicroinjection, cell fusion, microcell fusion, electroporation,electrofusion, projectile bombardment, calcium phosphate precipitation,site-specific targeting and other such methods. Cells particularlysuited for use with the artificial chromosomes include, but are notlimited to plant cells, particularly tomato, arabidopsis, and others,insect cells, including silk worm cells, insect larvae, fish, reptiles,amphibians, arachnids, mammalian cells, embryonic stem cells, embryosand cells for use in methods of genetic therapy, such as lymphocytesthat are used in methods of adoptive immunotherapy and nerve or neuralcells. Thus methods of producing gene products and transgenic animalsand plants are provided. Also provided are the resulting transgenicanimals and plants.

[0027] Exemplary cell lines that contain these chromosomes are alsoprovided.

[0028] Methods for preparing artificial chromosomes for particularspecies and for cloning centromeres are also provided. In particular, amethod for cloning a centromere from an animal or plant by preparing alibrary of DNA fragments that contain the genome of the plant or animal,introducing the each of the fragments into a mammalian satelliteartificial chromosome (SATAC) that contains a centromere from adifferent species, generally a mammal, from the selected plant oranimal, generally a non-mammal, and a selectable marker. The selectedplant or animal is one in which the mammalian species centromere doesnot function. Each of the SATACs is introduced into the cells, which aregrown under selective conditions, and cells with SATACs are identified.Such SATACS should contain a centromere encoded by the DNA from thelibrary.

[0029] Also provided are libraries in which the relatively largefragments of DNA are contained on artificial chromosomes.

[0030] Transgenic animals, invertebrates and vertebrates, plants andinsects, fish, reptiles, amphibians, arachnids and mammals are alsoprovided. Of particular interest are transgenic animals that expressgenes that confer resistance or reduce susceptibility to disease. Sincemultiple genes can be introduced on a MAC, a series of genes encoding anantigen can be introduced, which up expression will serve to immunize(in a manner similar to a multivalent vaccine) the host animal againstthe diseases for which exposure to the antigens provide immunity or someprotection.

[0031] Methods for cloning centromeres, such as mammalian centromeres,are also provided. In particular, in one embodiment, a library composedof fragments of the SATACs are cloned into YACs (yeast artificialchromosomes) that include a detectable marker, such as DNA encodingtyrosinase and then introduced into mammalian cells, such as albinomouse embryos. Mice produced from YACs that include a centromere thatfunctions in mammals will express the detectable marker. Thus, if albinomice will be pigmented or have regions of pigmentation.

DESCRIPTION OF THE DRAWINGS

[0032]FIG. 1 is a schematic drawing depicting formation of the MMCneo(the minichromosome) chromosome. A-G represents the successive eventsconsistent with observed data that would lead to the formation andstabilization of the minichromosome.

[0033]FIG. 2 shows a schematic summary of the manner in which theobserved new chromosomes would form, and the relationships among thedifferent de novo formed chromosomes. In particular, this figure shows aschematic drawing of the de novo chromosome formation initiated in thecentromeric region of mouse chromosome 7. (A) A single E-typeamplification in the centromeric region of chromosome 7 generates aneo-centromere linked to the integrated “foreign” DNA, and forms adicentric chromosome (FIG. 2-1). Multiple E-type amplification forms theλ neo-chromosome, which was derived from chromosome 7 and stabilized ina mouse-hamster hybrid cell line (FIGS. 2-2 and 2-6); (B) Specificbreakage between the centromeres of a dicentric chromosome 7 generates achromosome fragment with the neo-centromere, and a chromosome 7 withtraces of heterologous DNA at the end (FIG. 2-3); (C) Invertedduplication of the fragment bearing the neo-centromere results in theformation of a stable neo-minichromosome (FIG. 2-4); (D) Integration ofexogenous DNA into the heterologous DNA region of the formerly dicentricchromosome 7 initiates H-type amplification, and the formation of aheterochromatic arm. By capturing a euchromatic terminal segment, thisnew chromosome arm is stabilized in the form of the “sausage” chromosome(FIG. 2-5); (E) BrdU (5-bromodeoxyuridine), treatment and/or drugselection induce further H-type amplification, which results in theformation of an unstable gigachromosome (FIG. 2-7); (F) Repeated BrdUtreatments and/or drug selection induce further H-type amplificationincluding a centromere duplication, which leads to the formation ofanother heterochromatic chromosome arm. It is split off from thechromosome 7 by chromosome breakage, and by acquiring a terminalsegment, the stable megachromosome is formed (FIG. 2-8).

[0034]FIG. 3 Schematic diagram of the replicon structure and a scheme bywhich the megachromosome could be produced.

[0035]FIG. 4 sets forth the relationships among the some of theexemplary cell lines described herein.

[0036]FIG. 5 is diagram of the plasmid pTEMPUD.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0037] Definitions

[0038] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as is commonly understood by one of skillin the art to which this invention belongs. All patents and publicationsreferred to herein are incorporated by reference.

[0039] As used herein, a mammalian artificial chromosome (MAC) is pieceof DNA that can stably replicate and segregate alongside endogenouschromosomes. It has the capacity to accommodate and express heterologousgenes inserted therein. It is referred to as a mammalian artificialchromosome because it includes an active mammalian centromere. Plantartificial chromosomes and an insect artificial chromosomes refer tochromosomes that include plant and insect centromeres, respectively. Ahuman specific chromosome (HAC) refers to chromosomes that include humancentromeres, BUGACs refer to artificial insect chromosomes, and AVACsrefer to avian artificial chromosomes.

[0040] As used herein, stable maintenance of chromosomes, occurs when atleast about 85%, preferably 90%, more preferably 95%, of the cellsretain the chromosome. Stability is measured in the presence ofselective agent. Preferably these chromosomes are also maintained in theabsence of a selective agent. Stable chromosomes also retain theirstructure during cell culturing, suffering neither intrachromosomal norinterchromosomal rearrangements.

[0041] As used herein, growth under selective conditions, means growthof a cell under conditions that require expression of a selectablemarker for survival.

[0042] As used herein, euchromatin and heterochromatin have theirrecognized meanings, euchromatin refers to DNA that contains genes, andheterochromatin refers to chromatin that has been thought to beinactive. Highly repetitive DNA sequences (satellite DNA) are located inregions of centromeric heterochromatin (pericentric heterochromatin).Constitutive heterochromatin refers to heterochromatin that contains thehighly repetitive DNA and that is constitutively condensed.

[0043] As used herein, BrdU refers to 5-bromodeoxyuridine, which duringreplication is inserted in place of thymidine. BrdU is used as mutagen;it also inhibits condensation of metaphase chromosomes during celldivision.

[0044] As used herein, a dicentric chromosome is a chromosome thatcontains two centromeres. A multicentric chromosome contains more thantwo centromeres.

[0045] As used herein, a formerly dicentric chromosome is a chromosomethat is produced when a dicentric chromosome fragments and acquires newtelomeres so that two chromosomes, each having one of the centromeres,are produced. Each of the fragments, are replicable chromosomes. If oneof the chromosomes undergoes amplification of euchromatic DNA to producea full functionally chromosome that contains the heterologous DNA andprimarily (at least more than 50%) euchromatin, it is a minichromosome.The remaining chromosome is a formerly dicentric chromosome. If one ofthe chromosomes undergoes amplification, whereby heterochromatin(satellite DNA) is amplified, a euchromatic portion (or arm remains), itis referred to as a sausage chromosome. A chromosome that issubstantially all heterochromatin, except for portions of heterologousDNA, is called a SATAC. Such chromosomes (SATACs) can be produced fromsausage chromosomes by culturing the cell containing the sausagechromosome under conditions, such as BrdU treatment and/or growth underselective conditions, that destabilize the chromosome so that asatellite artificial chromosomes (SATAC) is produced. For purposesherein, it is understand that SATACs may not necessarily be produced inmultiple steps, but may appear after the initial introduction of theheterologous DNA and growth under selective conditions, or they mayappear after several cycles of growth under selective conditions andBrdU treatment.

[0046] As used herein an amplicon is the smallest repeated unit thatcontains heterologous DNA in the MACs provided herein. A megarepliconcontains at least one amplicon, an inverted repeat thereof, and amegareplicator. A megareplicator is a primary replication initiationsite.

[0047] As used herein, the minichromosome refers to a chromosome derivedfrom a dicentric chromosome (see, e.g., FIG. 1) that contains moreeuchromatic than heterochromatic DNA.

[0048] As used herein, a megachromosome refers to a chromosome that,except for introduced heterologous DNA is substantially composed ofheterochromatin. Megachromosomes are made of a tandem array of ampliconsthat contain two inverted megareplicons bordered by introducedheterologous DNA (see, e.g., FIG. 3 for a schematic drawing of amegachromosome). For purposes herein, a megachromosome is about 50 to400 Mb, generally about 250-400 Mb. Shorter variants, are also referredto as truncated megachromosomes (about 90 to 120 or 150 Mb), dwarfmegachromosomes (˜150-200 Mb) and cell lines, and micro-megachromosomes(˜60-90 Mb). For purposes herein, the term megachromosome refers to theoverall repeated structure based on a tandem array of repeatedchromosomal segments (amplicons) that contain two inverted megarepliconsbordered by any inserted heterologous DNA. The size will be specified.

[0049] As used herein, genetic therapy involves the transfer ofheterologous DNA to the certain cells, target cells, of an individualafflicted with a disorder for which such therapy is sought. The DNA isintroduced into the selected target cells in a manner such that theheterologous DNA is expressed and a product encoded thereby is produced.Alternatively, the heterologous DNA may in some manner mediateexpression of DNA that encodes the therapeutic product, it may encode aproduct, such as a peptide or RNA that in some manner mediates, directlyor indirectly, expression of a therapeutic product. Genetic therapy mayalso be used to introduce therapeutic compounds, such as TNF, that arenot normally produced in the host or that are not produced intherapeutically effective amounts or at a therapeutically useful time.The heterologous DNA encoding the therapeutic product may be modifiedprior to introduction into the cells of the afflicted host in order toenhance or otherwise alter the product or expression thereof.

[0050] As used herein, heterologous or foreign DNA and RNA are usedinterchangeably and refer to DNA or RNA that does not occur naturally aspart of the genome in which it is present or which is found in alocation or locations in the genome that differ from that in which itoccurs in nature. It is DNA or RNA that is not endogenous to the celland has been exogenously introduced into the cell. Examples ofheterologous DNA include, but are not limited to, DNA that encodes agene or gene(s) of interest, introduced for purposes of gene therapy orfor production of the encoded protein. Other examples of heterologousDNA include, but are not limited to, DNA that encodes traceable markerproteins, such as a protein that confers drug resistance, DNA thatencodes therapeutically effective substances, such as anti-canceragents, enzymes and hormones, and DNA that encodes other types ofproteins, such as antibodies. Antibodies that are encoded byheterologous DNA may be secreted or expressed on the surface of the cellin which the heterologous DNA has been introduced.

[0051] As used herein, a therapeutically effective product is a productthat is encoded by heterologous DNA that, upon introduction of the DNAinto a host, a product is expressed that effectively ameliorates oreliminates the symptoms, manifestations of an inherited or acquireddisease or that cures said disease.

[0052] As used herein, transgenic plants refer to plants in whichheterologous or foreign DNA is expressed or in which the expression of agene naturally present in the plant has been altered.

[0053] As used herein, operative linkage of heterologous DNA toregulatory and effector sequences of nucleotides, such as promoters,enhancers, transcriptional and translational stop sites, and othersignal sequences refers to the relationship between such DNA and suchsequences of nucleotides. For example, operative linkage of heterologousDNA to a promoter refers to the physical relationship between the DNAand the promoter such that the transcription of such DNA is initiatedfrom the promoter by an RNA polymerase that specifically recognizes,binds to and transcribes the DNA in reading frame.

[0054] As used herein, isolated, substantially pure DNA refers to DNAfragments purified according to standard techniques employed by thoseskilled in the art, such as that found in Maniatis et al. ((1982)Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.).

[0055] As used herein, expression refers to the process by which nucleicacid is transcribed into mRNA and translated into peptides,polypeptides, or proteins. If the nucleic acid is derived from genomicDNA, expression may, if an appropriate eukaryotic host cell or organismis selected, include splicing of the mRNA.

[0056] As used herein, vector or plasmid refers to discrete elementsthat are used to introduce heterologous DNA into cells for eitherexpression of the heterologous DNA or for replication of the clonedheterologous DNA. Selection and use of such vectors and plasmids arewell within the level of skill of the art.

[0057] As used herein, transformation/transfection refers to the processby which DNA or RNA is introduced into cells to for gene expression.Transfection refers to the taking up of an expression vector by a hostcell whether or not any coding sequences are in fact expressed. Numerousmethods of transfection are known to the ordinarily skilled artisan, forexample, by direct uptake using calcium phosphate (CaPO4; see, e.g.,Wigler et al. (1979) Proc. Natl. Acad. Sci. U.S.A. 76:1373-1376) andpolyethylene glycol (PEG)-mediated DNA uptake and electroporation.Successful transfection is generally recognized when any indication ofthe operation of this vector occurs within the host cell. Transformationmeans introducing DNA into an organism so that the DNA is replicable,either as an extrachromosomal element or by chromosomal integrant.

[0058] As used herein, injected refers to the microinjection (use of asmall syringe) of DNA into a cell.

[0059] As used herein, substantially homologous DNA refers to DNA thatincludes a sequence of nucleotides that is sufficiently similar toanother such sequence to form stable hybrids under specified conditions.

[0060] It is well known to those of skill in this art, that nucleic acidfragments with different sequences may, under the same conditions,hybridize detectably to the same “target” nucleic acid. Two nucleic acidfragments hybridize detectably, under stringent conditions over asufficiently long hybridization period, because one fragment contains asegment of at least about 14 nucleotides in a sequence which iscomplementary (or nearly complementary) to the sequence of at least onesegment in the other nucleic acid fragment. If the time during whichhybridization is allowed to occur is held constant, at a value duringwhich, under preselected stringency conditions, two nucleic acidfragments with exactly complementary base-pairing segments hybridizedetectably to each other, departures from exact complementarity can beintroduced into the base-pairing segments, and base-pairing willnonetheless occur to an extent sufficient to make hybridizationdetectable. As the departure from complementarity between thebase-pairing segments of two nucleic acids becomes larger, and asconditions of the hybridization become more stringent, the probabilitydecreases that the two segments will hybridize detectably to each other.

[0061] Two single-stranded nucleic acid segments have “substantially thesame sequence,” within the meaning of the present specification, if (a)both form a base-paired duplex with the same segment, and (b) themelting temperatures of said two duplexes in a solution of 0.5×SSPEdiffer by less than 10° C. If the segments being compared have the samenumber of bases, then to have “substantially the same sequence”, theywill typically differ in their sequences at fewer than 1 base in 10.Methods for determining melting temperatures of nucleic acid duplexesare well known (see, e.g., Meinkoth and Wahl (1984) Anal. Biochem.138:267-284 and references cited therein).

[0062] As used herein, a nucleic acid probe is a DNA or RNA fragmentthat includes a sufficient number of nucleotides to specificallyhybridize to DNA or RNA that includes identical or closely relatedsequences of nucleotides. A probe may contain any number of nucleotides,from as few as about 10 and as many as hundreds of thousands ofnucleotides. The conditions and protocols for such hybridizationreactions are well known to those of skill in the art as are the effectsof probe size, temperature, degree of mismatch, salt concentration andother parameters on the hybridization reaction. For example, the lowerthe temperature and higher the salt concentration at which thehybridization reaction is carried out, the greater the degree ofmismatch that may be present in the hybrid molecules.

[0063] To be used as an hybridization probe, the nucleic acid isgenerally rendered detectable by labelling it, with a detectable moietyor label, such as ³²P, ³H and ¹⁴C, or by other means, including chemicallabelling, such as by nick-translation in the presence of deoxyuridylatebiotinylated at the 5′-position of the uracil moiety. The resultingprobe includes the biotinylated uridylate in place of thymidylateresidues and can be detected (via the biotin moieties) by any of anumber of commercially available detection systems based on binding ofstreptavidin to the biotin. Such commercially available detectionsystems can be obtained, for example, from Enzo Biochemicals, Inc. (NewYork, N.Y.). Any other label known to those of skill in the art,including non-radioactive labels, may be used as long as it renders theprobes sufficiently detectable, which is a function of the sensitivityof the assay, the time available (for culturing cells, extracting DNA,and hybridization assays), the quantity of DNA or RNA available as asource of the probe, the particular label and the means used to detectthe label.

[0064] Once sequences with a sufficiently high degree of homology to theprobe are identified, they can readily be isolated by standardtechniques, which are described, for example, by Maniatis et al. ((1982)Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.).

[0065] As used herein, conditions under which DNA molecules form stablehybrids and are considered substantially homologous are such that theDNA molecules with at least about 60% complementarity form stablehybrids. Such DNA fragments are herein considered to be “substantiallyhomologous”. For example, DNA that encodes a particular protein issubstantially homologous to an other DNA fragment if the DNA formsstable hybrids such that the sequences of the fragments are at leastabout 60% complementary and if a protein encoded by the DNA retains itsactivity.

[0066] For purposes herein, the following stringency conditions aredefined:

[0067] 1) high stringency: 0.1×SSPE, 0.1% SDS, 65° C.

[0068] 2) medium stringency: 0.2×SSPE, 0.1% SDS, 50° C.

[0069] 3) low stringency: 1.0×SSPE, 0.1% SDS, 50° C.

[0070] or any combination of salt and temperature and other reagentsthat result in selection of the same degree of mismatch or matching.

[0071] As used herein, immunoprotective refers to the ability of avaccine or exposure to an antigen or immunity-inducing agent to conferupon a host to whom the vaccine or antigen is administered or introducedthe ability to resist infection by a disease causing pathogen or to havereduced symptoms. The selected antigen is typically an antigen that ispresented by the pathogen.

[0072] As used herein, all assays and procedures, such as hybridizationreactions and antibody-antigen reactions, unless otherwise specified,are conducted under conditions recognized by those of skill in the artas standard conditions.

[0073] A. Preparation of Cell Lines Containing MACs

[0074] The methods, cells and MACs provided herein are produced byvirtue of the discovery of the existence of a higher-order replicationunit (megareplicon) of the centromeric region. This megareplicon isdelimited by a primary replication initiation site (megareplicator), andappears to facilitate replication of the centromeric heterochromatin,and most likely, centromeres. Integration of heterologous DNA into themegareplicator region or in close proximity thereto, initiates alarge-scale amplification of megabase-size chromosomal segments, whichleads to de novo chromosome formation.

[0075] Cell lines containing MACs can be prepared using cells,preferably a stable cell line, transforming it with a heterologous DNAfragment that encodes a selectable marker, culturing under selectiveconditions, and identifying cells that have a dicentric chromosome.These cells can then be manipulated as described herein to produce theminichromosomes and other MACs, particularly the heterochromatic SATACsas described herein.

[0076] Development of a dicentric chromosome appears to requireintegration of the heterologous DNA in the pericentric heterochromatin.Thus, the probability of incorporation can be increased by includingDNA, such as satellite DNA, in the heterologous fragment that encodesthe selectable marker. The resulting cell lines can then be treated asthe exemplified cells herein to produce cell in which the dicentricchromosome has fragmented and to introduce additional selective markersinto the dicentric chromosome, whereby amplification of the pericentricheterochromatin will produce the heterochromatic chromosomes. Thefollowing discussion is with reference to the EC3/7 line and use ofresulting cells. The same procedures can be applied and to any othercells, particularly cell lines to prepare create SATACs and euchromaticminichromosomes.

[0077] 1. Formation of De Novo Chromosomes

[0078] De novo centromere formation in a transformed mouseLMTK-fibroblast cell line (EC3/7) after cointegration of λ constructs(λCM8 and λgtWESneo) carrying human and bacterial DNA (Hadlaczky et al.(1991) Proc. Natl. Acad. Sci. U.S.A. 88:8106-8110 and U.S. applicationSer. No. 08/375,271) has been shown. The integration of the“heterologous” human and phage DNA, and the subsequent amplification ofmouse and heterologous DNA that led to the formation of a dicentricchromosome, occurred at the centromeric region of the short arm of amouse chromosome. By G-banding this chromosome was identified as mousechromosome 7. Because of the presence of two functionally activecentromeres on the same chromosome, regular breakages occur between thecentromeres. Such specific chromosome breakages gave rise to theappearance (in approximately 10% of the cells) of a chromosome fragmentcarrying the neo-centromere. From the EC3/7 cell line (see, U.S. Pat.No. 5,288,625, deposited at the European Collection of Animal cellCulture (hereinafter ECACC) under accession no. 90051001); see, alsoHadlaczky et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88:8106-8110, andU.S. application Ser. No. 08/375,271 and the corresponding publishedEuropean application EP 0 473 253) carrying either the dicentricchromosome or a chromosome fragment with the neo-centromere, twosublines (EC3/7C5 and EC3/7C6) were selected by repeated single-cellcloning. In these cell lines, the neo-centromere was found exclusivelyon a minichromosome (neo-minichromosome)), while the formerly dicentricchromosome carried traces of “heterologous” DNA.

[0079] It has now been discovered that integration of DNA encoding aselectable marker in the heterochromatic region of the centromere led toformation of the dicentric chromosome.

[0080] 2. The Neo-Minichromosome

[0081] The chromosome breakage in the EC3/7 cells, which separates theneo-centromere from the mouse chromosome, occurred in the G-bandpositive “heterologous” DNA region. This is supported by the observationof traces of λ and human DNA sequences at the broken end of the formerlydicentric chromosome. Comparing the G-band pattern of the chromosomefragment carrying the neo-centromere with that of the stableneo-minichromosome, it is apparent that the neo-minichromosome is aninverted duplicate of the chromosome fragment that bears theneo-centromere. This is supported by the observation that although theneo-minichromosome carries only one functional centromere, both ends ofthe minichromosome are heterochromatic, and mouse satellite DNAsequences were found in these heterochromatic regions by in situhybridization.

[0082] Mouse cells containing the minichromosome, which is composed ofmultiple repeats of the heterologous DNA, which in the exemplifiedembodiment is lambda DNA and neo DNA, can be used as recipient cells incell transformation. Donor DNA, such as selected heterologous DNA linkedto a second selectable marker, such as hygromycin resistance (hyg), canbe introduced into the mouse cells and integrated into theminichromosomes by homologous recombination of lambda DNA in the donorDNA with that in the minichromosomes. Integration is verified by in situhybridization and Southern blot analyses. Transcription and translationof the heterologous DNA is confirmed by primer extension and immunoblotanalyses.

[0083] For example, DNA has been targeted into the λ-neo minichromosomein EC3/7C5 cells using a lambda DNA-containing construct (pNem1ruc) thatalso contains DNA encoding hygromycin resistance and the Renillaluciferase gene linked to a promoter, such as the cytomegalovirus (CMV)early promoter, and the bacterial neo encoding DNA. Integration of thedonor DNA into the chromosome in selected cells (designated PHN4) wasconfirmed by nucleic acid amplification (PCR) and in situ hybridization.Events that would produce a neo-minichromosome are depicted in FIG. 1.

[0084] The resulting engineered minichromosome that contains theheterologous DNA can then transferred by cell fusion into a recipientcell line, such as Chinese hamster kidney cells (CHO) and correctexpression of the heterologous DNA can be verified. Following productionof the cells, metaphase chromosomes are obtained, such as by addition ofcolchicine, and the chromosomes purified by addition of AT and GCspecific dyes on a dual laser beam based cell sorter. Preparativeamounts of chromosomes (2-3 mls of 10⁶ chromosomes/ml) at a purity of95% or higher can be obtained. The resulting chromosomes are used fordelivery to cells by methods, such as microinjection, liposome packagedtransfer.

[0085] Thus, the neo-minichromosome is stably maintained in cells,replicates autonomously, and permits the persistent long-term expressionof neo gene under non-selective culture conditions. It also containsmegabases of heterologous known DNA (lambda DNA in the exemplifiedembodiments) that serves as target sites form homologous recombinationand integration of DNA of interest. The neo-minichromosome is, thus, avector for genetic engineering of cells.

[0086] The methods herein provide means to induce the events that led toformation of the neo-minichromosome by introducing heterologous DNA witha selective marker (preferably a dominant selectable marker) andculturing under selective conditions. As a result, cells that contain adicentric chromosome or fragments thereof produced by amplification,will be produced. Cells with the dicentric chromosome can then betreated to destabilize the genome with agents, such as BrdU and/orculturing under selective conditions, resulting in cells in which thedicentric chromosome has formed two chromosomes, a so-calledminichromosome, and a formerly dicentric chromosome that has typicallyundergone amplification in the heterochromatin where the heterologousDNA has integrated to produce a generally a SATAC or a sausagechromosome (discussed below). These cells can be fused with other cellsto separate the minichromosome from the formerly dicentric chromosomeinto different cells so that each type of MAC can be manipulatedseparately.

[0087] 3. Preparation of SATACs

[0088] An Exemplary protocol for preparation of SATACs is illustrated inFIG. 2 (particularly C, D and F) and FIG. 4 (see, also the EXAMPLES,particularly EXAMPLES 4-7).

[0089] To prepare a SATAC, the starting materials are a cell, preferablya stable cell line, such as a fibroblast cell line, and a DNA fragmentthat includes DNA that encodes a selective marker. To insure integrationof the DNA fragment in the heterochromatin, it is preferable to startwith DNA that will be targeted to the pericentric heterochromatic regionof the chromosome, such as λCM8 and vectors provided herein, such aspTEMPUD (FIG. 5) that include satellite DNA. After introduction of theDNA, the cells are grown under selective conditions. The resulting cellsare examined and any that have dicentric chromosomes (or heterochromaticchromosomes or sausage chromosomes or other such structure (see, FIGS.2C, 2D, 2E and 2F) are selected.

[0090] In particular, if a cell with a dicentric chromosome is selected,it can be grown under selective conditions, or, preferably, additionalDNA encoding a second selectable marker is introduced, and the cellsgrown under conditions selective for the second marker. The resultingcells should include chromosomes that have structures similar to thosedepicted in FIGS. 2D, 2E, 2F. Cells with a structure, such as thesausage chromosome, FIG. 2D, can be selected and fused with a secondcell line to eliminate other chromosomes that are not of interest. Ifdesired cells with other chromosomes can be selected and treated asdescribed herein. If a cell with a sausage chromosome is selected, it istreated with an agent, such as BrdU, that destabilizes the chromosome sothat the heterochromatic arm forms a chromosome that is substantiallyheterochromatin (megachromosome, see, FIG. 2F). Structures such as thegigachromsome in which the heterochromatic arm has amplified but notbroken off from the euchromatic arm, will also be observed. Themegachromosome is a stable chromosome. Further manipulation, such asfusions and growth in selective conditions and/or BrdU treatment orother such treatment, can lead to fragmentation of the megachromosome toform smaller chromosomes that have the amplicon as the basic repeatingunit.

[0091] The megachromosome can be further fragmented in vivo using achromosome fragmentation vector, such as pTEMPUD (see, FIG. 5 andEXAMPLE 12) to ultimately produce a chromosome that comprises thesmallest stable replicable unit, about 15 Mb-50 Mb, containing two tofour megareplicons.

[0092] Thus, the stable chromosomes formed de novo that originate fromthe short arm of mouse chromosome 7 have been analyzed. This chromosomeregion shows a capacity for amplification of large chromosome segments,and promotes de novo chromosome formation. Large-scale amplification atthe same chromosome region leads to the formation of dicentric andmulticentric chromosomes, a minichromosome, the 150-200 Mb size λneo-chromosome, the “sausage” chromosome, the 500-1000 Mbgigachromosome, and the stable 250-400 Mb megachromosome.

[0093] A clear segmentation is observed along the arms of themegachromosome, and analyses show that the building units of thischromosome are amplicons of ˜30 Mb composed of mouse major satellite DNAwith the integrated “foreign” DNA sequences at both ends. The ˜30 Mbamplicons are composed of two ˜15 Mb inverted doublets of ˜7.5 Mb mousemajor satellite DNA blocks, which are separated from each other by anarrow band of non-satellite sequences (see, e.g., FIG. 3). The widernon-satellite regions at the amplicon borders contain integrated,exogenous (heterologous) DNA, while the narrow bands of non-satelliteDNA sequences within the amplicons are integral parts of the pericentricheterochromatin of mouse chromosomes. These results indicate that the˜7.5 Mb blocks flanked by non-satellite DNA are the building units ofthe pericentric heterochromatin of mouse chromosomes, and the 15 Mb sizepericentric regions of mouse chromosomes contain two ˜7.5 Mb units.

[0094] Apart from the euchromatic terminal segments, the wholemegachromosome is heterochromatic, and has structural homogeneity.Therefore, this large chromosome offers a unique possibility forobtaining information about the amplification process, and for analyzingsome basic characteristics of the pericentric constitutiveheterochromatin, as vector for heterologous DNA, and as target forfurther fragmentation.

[0095] As shown herein, this phenomenon is generalizable and can beobserved with other chromosomes. Also, although these de novo formedchromosome segments and chromosomes appear different, there are,similarities that indicate that a similar amplification mechanism playsa role in their formation: (i) in each case, the amplification isinitiated in the centromeric region of the mouse chromosomes and large(Mb size) amplicons are formed; (ii) mouse major satellite DNA sequencesare constant constituents of the amplicons, either by providing the bulkof the heterochromatic amplicons (H-type amplification), or by borderingthe euchromatic amplicons (E-type amplification); (iii) formation ofinverted segments can be demonstrated in the λ neo-chromosome andmegachromosome; (iv) chromosome arms and chromosomes formed by theamplification are stable and functional.

[0096] The presence of inverted chromosome segments seems to be a commonphenomenon in the chromosomes formed de novo at the centromeric regionof mouse chromosome 7. During the formation of the neo-minichromosome,the event leading to the stabilization of the distal segment of mousechromosome 7 that bears the neo-centromere may have been the formationof its inverted duplicate. Amplicons of the megachromosome are inverteddoublets of ˜7.5 Mb mouse major satellite DNA blocks.

[0097] 4. Cell Lines

[0098] Cell lines that contain MACs, such as the minichromosome, theλ-neo chromosome, and the SATACs are provided herein or can be producedby the methods herein. Such cell lines provide a convenient source ofthese chromosomes and can be manipulated, such as by cell fusion orproduction of microcells for fusion with selected cell lines, to deliverthe chromosome of interest into hybrid cell lines. Exemplary cell linesare described herein and some have been deposited with the ECACC.

[0099] a. EC3/7C5 and EC3/7C6

[0100] Two cell lines EC3/7C5 and EC3/7C6 produced by single cellcloning of EC3/7 were examined. For exemplary purposes EC3/7C5 has beendeposited with the ECACC. These cell lines contain a minichromosome andthe formerly dicentric chromosome from EC3/7. The stable minichromosomesin cell lines (EC3/7C5 and EC3/7C6) appear to be virtually identical andthey seem to be a duplicated derivatives of the ˜10-15 Mb “broken-off”fragment of the dicentric chromosome. Their identical size in theseindependently generated cell lines might indicate that ˜20-30 Mb is theminimal or close to the minimal physical size for a stableminichromosome.

[0101] b. TF1004G/19

[0102] Introduction of additional heterologous DNA, including a secondselectable marker hygromycin and also a detectable markerβ-galactosidase, into the EC3/7C5 cell line and growth under selectiveconditions produced cells designated TF1004G/19. In particular, thiscell line was produced from the EC3/7C5 cell line by cotransfection withplasmids pH132, which contains an anti-HIV ribozyme, and hygromycinresistance gene, pCH110 (encodes β-galactosidase) and λ phage (λcl 875Sam 7) DNA and selection with hygromycin B.

[0103] Detailed analysis of TF1004G/19 cell line by in situhybridization with lambda phage and plasmid DNA sequences revealed theformation of the sausage chromosome. The formerly dicentric chromosomeof EC3/7C5 cell translocated to the end of another acrocentricchromosome. The heterologous DNA integrated into the pericentricheterochromatin of formerly dicentric chromosome and is amplifiedseveral times with megabases of mouse pericentric heterochromaticsatellite DNA sequences (FIG. 2D)) forming the “sausage” chromosome.Subsequently the acrocentric mouse chromosome was substituted by aeuchromatic telomere.

[0104] In situ hybridization with biotin labeled subfragments of thehygromycin resistance and β-galactosidase genes resulted inhybridization signal only in the heterochromatic arm of the sausagechromosome, indicating that in TF1004G/19 transformant cells these genesare localized in the pericentric heterochromatin. A high level of geneexpression, however, was detected.

[0105] In general, heterochromatin has a silencing effect in Drosophila,yeast and on the HSV-tk gene introduced into satellite DNA at mousecentromere. Thus, it was of interest to study the TF 1004G/19transformant cell line in to confirm that gene expression (of the β-gal)was indeed localized in the heterochromatin contrary to recognizeddogma.

[0106] For this purpose, subclones of TF1004G/19 containing a differenta sausage chromosome (see FIG. 2D) by single cell cloning wereestablished. Southern DNA hybridization with subfragments of hygromycinresistance and β-galactosidase genes showed close correlation to theintensity of hybridization and the length of sausage chromosome. Thisfinding supports the conclusion that these genes are localized in theheterochromatic arm of the sausage chromosome.

[0107] (1) TF1004G-19C5

[0108] TF1004G-19C5 is a mouse LMTK⁻ fibroblast cell line containingneo-minchromosomes and stable “sausage” chromosomes. It is a subclone ofTF1004G/19. It has been deposited as an exemplary cell line andexemplary source of a sausage chromosome. Subsequent fusion of this cellline with CHO K20 cells and selection with hygromycin and hat resultedin hybrid cells that carry the sausage chromosome and/or theneo-minichromosome. BrdU treatment, single cell cloning and selectionwith G418 and/or hygromycin produced various cells that carrychromosomes of interest, including G3D5.

[0109] (2) Other Subclones

[0110] G3D5, which has been deposited is a mouse hamster hybrid cellline that carries the neo-minichromosome and the megachromosome. H1D3 isa subclone thereof that carries the megachromosome. Fusion of this cellline with the CD4⁺ Hela cell line and also DNA encoding an additionalselection gene, neo, produced cells that carry the megachromosome aswell as a human chromosome that carries CD4neo (H1D3 cells). FurtherBrdU treatment and single cell cloning produced cell lines, such as 1B3that include cells with a truncated megachromosome.

[0111] 5. DNA Constructs Used to Transform the Cells

[0112] Heterologous DNA can be introduced into the cells by transfectionor other suitable method at any stage during preparation of thechromosomes (see, e.g., FIG. 4). In general integration of such DNA isassured by relying on site directed integration, such as by inclusion ofλ-DNA for the exemplified chromosomes and also an additional selectivemarker gene. For example, cells with a MAC, such as the minichromosomeor a SATAC can be cotransfected with a plasmid encoding the desiredheterologous DNA, such as HIV ribozyme, cystic fibrosis gene, and asecond selectable marker, such as hygromycin resistance. Selection iseffected with the agent that selects for the new selectable marker cellscontaining chromosomes that include the DNA in the MAC are identified.Fusion with a second cell line can provide a means to produce cell linesthat contain one particular type of chromosomal structure or MAC.

[0113] Various vectors for this purpose are provided herein (see,Examples) and others can be readily constructed. The vectors shouldinclude DNA that will target the DNA to the MAC, a selectable marker andthe selected heterologous gene of interest. Based on the disclosureherein and the knowledge of the skilled artisan, one of skill canconstruct such vectors.

[0114] Of particular interest herein is the vector pTEMPUD andderivatives thereof that can target DNA into the heterochromatic regionof selected chromosomes. These vectors can also serve as fragmentationvectors (see, e.g., Example 12).

[0115] Heterologous genes of interest include any gene that encodes atherapeutic gene and DNA encoding gene products of interest. These genesand DNA include, but are not limited to: the cystic fibrosis gene (CF)cystic fibrosis transmembrane regulator (CFTR) (see, e.g., U.S. Pat. No.5,240,846; Rosenfeld et al. (1992) Cell 68:143-155; Hyde et al. (1993)Nature 362: 250-255; Kerem et al. (1989) Science 245:1073-1080; Riordanet al. (1989) Science 245:1066-1072; Rommens et al. (1989) Science245:1059-1065; Osborne et al. (1991) Am. J. Hum. Genetics 48:6089-6122;White et al. (1990) Nature 344:665-667; Dean et al. (1990) Cell61:863-870; Erlich et al. (1991) Science 252:1643; and U.S. Pat. Nos.5,453,357, 5,449,604, 5,434,086, and 5,240,846, which provides aretroviral vector encoding the normal CFTR gene).

[0116] B. Isolation of Artificial Chromosomes

[0117] The MACs provided herein can be isolated by any suitable methodknown to those of skill in the art. Also, a method is provided hereinfor effecting substantial purification. SATACs have been isolated byfluorescence activated cell sorting (FACS). This method takes advantageof the nucleotide base content of the SATACs, which by virtue of theirheterochromatic content will differ from any other chromosomes in acell. In particular, metaphase chromosomes are isolated and stained withbase specific dyes, such as Hoechst 33258 and chromocycin A3.Fluorescence activated cell sorting will separate the SATACs from thegenomic chromosomes. A dual-laser cell sorter (FACStar Plus and FAXStarVantage Becton Dickinson Immunocytometry System) in which two laserswere set to excite the dyes separately, allowed a bivariate analysis ofthe chromosomes by size and base-pair composition. Cells containing suchSATACs can be similarly sorted.

[0118] C. Introduction of Artificial Chromosomes into Cells, Tissues,Animals and Plants

[0119] Suitable hosts for introduction of the MACs provided herein,include but are not limited to any animal or plant, cell or tissuethereof, including, but not limited to: mammals, birds, reptiles,amphibians, insects, fish, arachnids, tobacco, tomato, wheat, monocots,dicots and algae. The MACs may be introduced by cell fusion or microcellfusion or subsequent to isolation by any method known to those of skillin this art, including but not limited to: direct DNA transfer,electroporation, lipofection, liposomes, microprojectile bombardment,microinjection and any other suitable method.

[0120] Other methods for introducing DNA into cells, include nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells. Polycations, such as polybrene and polyornithine, may also beused. For various techniques for transforming mammalian cells, see e.g.,Keown et al. Methods in Enzymology (1990) Vol. 185, pp. 527-537; andMansour et al. (1988) Nature 336:348-352.

[0121] DNA may be introduced by direct DNA transformation;microinjection in cells or embryos, protoplast regeneration for plants,electroporation, microprojectile gun and other such methods (see, e.g.,Weissbach et al. (1988) Methods for Plant Molecular Biology, AcademicPress, N.Y., Section VIII, pp. 421-463; Grierson et al. (1988) PlantMolecular Biology, 2d Ed., Blackie, London, Ch. 7-9; see, also U.S. Pat.Nos. 5,491,075; 5,482,928; and 5,424,409; see, also, e.g., U.S. Pat. No.5,470,708, which describes particle-mediated transformation of mammalianunattached cells).

[0122] For example, isolated purified artificial chromosomes can beinjected into an embryonic cell line such as a human kidney primaryembryonic cell line (ATCC CRL 1573) or embryonic stem cells (see, e.g.,Hogan et al. (1994) Manipulating the Mouse Embryo, A :Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., see,especially, pages 255-264 and Appendix 3). Preferably the chromosomesare introduced by microinjection, using a system such as the Eppendorfautomated microinjection system, and grown under selective conditions,such as hygromycin B or neomycin resistance.

[0123] 1. Methods for Introduction of Chromosomes into Hosts

[0124] Depending on the host cell used, transformation is done usingstandard techniques appropriate to such cells. These methods includeany, including those described herein, known to those of skill in theart.

[0125] a. DNA Uptake

[0126] For mammalian cells without such cell walls, the calciumphosphate precipitation method (see, e.g., Graham et al. (1978) Virology52:456-457 is often preferred. DNA uptake can be accomplished by DNAalone or in the presence of polyethylene glycol (PEG-mediated genetransfer), which is a fusion agent, with plant protoplasts or by anyvariations of such methods known to those of skill in the art (see, etal. U.S. Pat. No. 4,684,611).

[0127] A commonly used approach for gene transfer in land plantsinvolves the direct introduction of purified DNA into protoplasts. Thethree basic methods for direct gene transfer include: 1) polyethyleneglycol (PEG)-mediated DNA uptake, 2) electroporation-mediated DNA uptakeand 3) microinjection. In addition, plants may be transformed usingultrasound treatment (see, e.g., International PCT application No. WO91/00358).

[0128] b. Electroporation

[0129] Electroporation, which involves providing high-voltage electricalpulses to a solution containing a mixture of protoplasts and foreign DNAto create reversible pores in the membranes of plant protoplasts as wellas other cells. Electroporation is generally used for prokaryotes orother cells, such as plants that contain substantial cell-wall barriers.Methods for effecting electroporation are well known (see, e.g., U.S.Pat. Nos. 4,784,737, 5,501,967, 5,501,662, 5,019,034, 5,503,999; see,also Fromm et al. (1985) Proc. Natl. Acad. Sci. U.S.A. 82:5824-5828).

[0130] For example, electroporation is often used for transformation ofplants (see, e.g., Ag Biotechnology News, Vol. 7 p. 3 and 17(September/October 1990)). In this technique, plant protoplasts areelectroporated in the presence of the DNA of interest that also includesa phenotypic marker. Electrical impulses of high field strengthreversibly permeabilize biomembranes allowing the introduction of theplasmids. Electroporated plant protoplasts reform the cell wall, divide,and form plant callus. Transformed plant cells will be identified byvirtue of the expressed phenotypic marker. The exogenous DNA may beadded to the protoplasts in any form such as, for example, naked linear,circular or supercoiled DNA, DNA encapsulated in liposomes, DNA inspheroplasts, DNA in other plant protoplasts, DNA complexed with salts,and other methods.

[0131] C. Microcells

[0132] The chromosomes can be transferred by preparing microcellscontaining an artificial chromosome and then fusing with selected targetcells. Methods for such preparation and fusion or microcells are wellknown (see, e.g., U.S. Pat. Nos. 5,240,840, 4,806,476, 5,298,429,Fournier (1981) Proc. Natl. Acad. Sci. U.S.A. 78:6349-6353; and Lambertet al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88:5907-59).

[0133] 2. Hosts

[0134] Suitable host include any host known to be useful forintroduction and expression of heterologous DNA. Of particular interestherein, animal and plant cells and tissues, including, but not limitedto insect cells and larvae, plants, and animals, particularly transgenicanimals, and animal cells. Other hosts include, but are not limited tomammals, birds, reptiles, amphibians, insects, fish, arachnids, tobacco,tomato, wheat, monocots, dicots and algae, and any host into whichintroduction of heterologous DNA is desired. Such introduction can beeffected using the MACs provided herein, or, if necessary by using theMACs provided herein to identify species-specific centromeres and/orfunctional chromosomal units and then using the resulting centromeres orchromosomal units as artificial chromosomes, or alternatively, using themethods exemplified herein for production of MACs to producespecies-specific artificial chromosomes.

[0135] a. Introduction of DNA into Embryos for Production of TransgenicAnimals and Introduction of DNA into Animal Cells

[0136] Transgenic animals can be produced by introducing exogenousgenetic material into a pronucleus of a mammalian zygote bymicroinjection (see, e.g., U.S. Pat. Nos. 4,873,191 and 5,354,674; see,also, International PCT application No. WO95/14769, which is based onU.S. application Ser. No. 08/159,084). The zygote is capable ofdevelopment into a mammal. The embryo or zygote is transplanted into ahost female uterus and allowed to develop. Detailed protocols andexamples are set forth below.

[0137] DNA can be introduced into animal cells using any knownprocedure, including, but not limited to: direct uptake, incubation withpolyethylene glycol (PEG), microinjection, electroporation, lipofection,cell fusion, microcell fusion, particle bombardment, includingmicroprojectile bombardment (see, e.g., U.S. Pat. No. 5,470,708, whichprovides a method for transforming unattached mammalian cells viaparticle bombardment), and any other such method. For example, thetransfer of plasmid DNA in liposomes directly to human cells in situ hasbeen approved by the FDA for use in humans (see, e.g., Nabel, et al.(1990) Science 249:1285-1288 and U.S. Pat. No. 5,461,032).

[0138] b. Introduction of Heterologous DNA into Plants.

[0139] Numerous methods for producing or developing transgenic plantsare available to those of skill in the art. The method used is primarilya function of the species of plant. These methods include, but are notlimited to: direct transfer of DNA by processes, such as PEG-induced DNAuptake, protoplast fusion, microinjection, electroporation, andmicroprojectile bombardment (see, e.g., Uchimiya et al. (1989) J. ofBiotech. 12: 1-20 for a review of such procedures, see, also, e.g., U.S.Pat. Nos. 5,436,392 and 5,489,520 and many others). For purposes herein,when introducing a MAC, microinjection and protoplast fusion arepreferred

[0140] Plant species, including tobacco, rice, maize, rye, soybean,Brassica napus, cotton, lettuce, potato and tomato, have been used toproduce transgenic plants. Tobacco and other species, such as petunias,often serve as experimental models in which the methods have beendeveloped and the genes first introduced and expressed.

[0141] DNA uptake can be accomplished by DNA alone or in the presence ofPEG, which is a fusion agent, with plant protoplasts or by anyvariations of such methods known to those of skill in the art (see,e.g., U.S. Pat. No. 4,684,611 to Schilperoot et al.). Electroporation,which involves high-voltage electrical pulses to a solution containing amixture of protoplasts and foreign DNA to create reversible pores, hasbeen used, for example, to successfully introduce foreign genes intorice and Brassica napus. Microinjection of DNA into plant cells,including cultured cells and cells in intact plant organs and embryoidsin tissue culture and microprojectile bombardment (acceleration of smallhigh density particles, which contain the DNA, to high velocity with aparticle gun apparatus, which forces the particles to penetrate plantcell walls and membranes) have also been used. All plant cells intowhich DNA can be introduced and that can be regenerated from thetransformed cells can be used to produce transformed whole plants whichcontain the transferred chromosome. The particular protocol and meansfor introduction of the DNA into the plant host may need to be adaptedor refined to suit the particular plant species or cultivar.

[0142] C. Insect Cells

[0143] Insects are useful hosts for introduction of artificialchromosomes for numerous reasons, including, but not limited to: (a)amplification of genes encoding useful proteins can be accomplished inthe artificial mammalian chromosome to obtain higher protein yields ininsect cells; (b) insect cells support required post translationalmodifications, such as glycosylation and phosphorylation, that can berequired for protein biological functioning; (c) insect cells do notsupport mammalian viruses, and, thus, eliminate the problem ofcross-contamination of products with such infectious agents; (d) thistechnology circumvents traditional recombinant baculovirus systems forproduction of nutritional, industrial or medicinal proteins in insectcell systems; (e) the low temperature optimum for insect cell growth(28° C.) permits reduced energy cost of production; (f) serum freegrowth medium for insect cells permits lower production costs; (g)artificial chromosome-containing cells can be stored indefinitely at lowtemperature; and (h) insect larvae will be biological factories forproduction of nutritional, medicinal or industrial proteins bymicroinjection of fertilized insect eggs (see, e.g., Joy et al. (1991)Current Science 66:145-150, which provides a method for microinjectingheterologous DNA into Bombyx mori eggs).

[0144] Either MACs or insect-specific (BUGACs) will used to introducegenes into insects. As described in the Examples, it appears that MACswill function in insects to direct expression of heterologous DNAcontained thereon.

[0145] Insect host cells include, but are not limited to, hosts such asSpodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedesalbopictus (mosquito), Drosophila melanogaster (fruitfly), Bombyx mori(silkworm) and Trichoplusia ni (cabbage looper). Effort haves beendirected toward propagation of insect cells in culture. Such effortshave focused on the fall armyworm, Spodoptera frugiperda. The cell lineshave been developed also from other insects such as the cabbage looper,trichoplusia ni and the silkworm, Bombyx mori. It has also beensuggested that analogous cell lines can be created using the tobaccohornworm, or Manduca sexta. To introduce DNA into an insect, it shouldbe introduced into the larvae, and allowed to proliferate, and then thehemolymph recovered from the larvae so that the proteins can be isolatedtherefrom. The preferred method herein is microinjection (see, e.g.,Tamura et al. (1991) Bio Ind. 8:26-31; Nikolaev et al. (1989) Mol. Biol.(Moscow) 23:1177-87; and methods exemplified and discussed herein).

[0146] D. Applications for and Uses of Artificial Chromosomes

[0147] Artificial chromosomes provide convenient and useful vectors, andin some instances the only vectors for introduction of heterologousgenes into hosts. Virtually any gene of interest is amenable tointroduction into a host via an artificial chromosomes. Such genesinclude, but are not limited to genes that encode receptors, cytokines,enzymes, proteases, hormones, growth factors, tumor suppressor genes,therapeutic products and multigene pathways.

[0148] The artificial chromosomes provided herein will be used inmethods of protein and gene product production, particularly usinginsects as host cells for production of such products. They are alsointended for use in methods of gene therapy, and in for production oftransgenic plants and animals (discussed above, below and in theEXAMPLES).

[0149] 1. Gene Therapy

[0150] Any therapeutic gene product or product of a multigene pathwaymay be introduced into a host animal, such as a human, or into a targetcell line for introduction into an animal, for therapeutic purposes.Such therapeutic purposes include, genetic therapy to cure or to providegene products that are missing or defective, to deliver agents, such asanti-tumor agents, to targeted cells or to an animal, and to providegene products that will confer resistance or reduce susceptibility to apathogen or ameliorate symptoms of a disease or disorder. The followingare some exemplary genes and gene products. Such exemplification is notintended to be limiting.

[0151] a. Anti-HIV Ribozymes

[0152] As exemplified below, DNA encoding anti-HIV ribozymes can beintroduced and expressed using MACs, including the euchromatin-basedminichromosomes and the SATACs. These MACs can be used to make atransgenic mouse with that express a ribozyme and, thus, serve as amodel for testing the activity of such ribozymes or from whichribozyme-producing cell lines can be made. Also, introduction of a MACinto human cells that encodes an anti-HIV ribozyme will serve astreatment for HIV infection.

[0153] b. Tumor Suppressor Genes

[0154] Tumor suppressor genes are genes that, in their wild-typealleles, express proteins that suppress abnormal cellular proliferation.When the gene coding for a tumor suppressor protein is mutated ordeleted, the resulting mutant protein or the complete lack of tumorsuppressor protein expression may fail to correctly regulate cellularproliferation, and abnormal cellular proliferation may take place,particularly if there is already existing damage to the cellularregulatory mechanism. A number of well-studied human tumors and tumorcell lines have been shown to have missing or nonfunctional tumorsuppressor genes.

[0155] Examples of tumor suppression genes include, but are not limitedto, the retinoblastoma susceptibility gene or RB gene, the p53 gene, thedeleted in colon carcinoma (DCC) gene and the neurofibromatosis type 1(NF-1) tumor suppressor gene (see, e.g., U.S. Pat. No. 5,496,731;Weinberg et al. (1991) 254:1138-1146). Loss of function or inactivationof tumor suppressor genes may play a central role in the initiationand/or progression of a significant number of human cancers.

[0156] The p53 Gene

[0157] Somatic cell mutations of the p53 gene are said to be the mostfrequently mutated gene in human cancer (see, e.g., Weinberg et al.(1991) Science 254:1138-1146). The normal or wild-type p53 gene is anegative regulator of cell growth, which, when damaged, favors celltransformation. The p53 expression product is found in the nucleus,where it may act in parallel with or cooperatively with other geneproducts. Tumor cell lines in which p53 has been deleted have beensuccessfully treated with wild-type p53 vector to reduce tumorigenicity(see, Baker et al. (1990) Science 249:912-915).

[0158] DNA encoding the p53 gene and plasmids containing this DNA arewell known (see, e.g., U.S. Pat. No. 5,260,191; see, also Chen et al.(1990) Science 250:1576; Farrel et al. (1991) EMBO J. 10:2879-2887,plasmids containing the gene are available from the ATCC, and thesequence is in the GenBank Database, accession nos. X54156, X60020,M14695, M16494, K03199).

[0159] c. The CFTR Gene

[0160] Cystic fibrosis (CF) is an autosomal recessive disease thataffects epithelia of the airways, sweat glands, pancreas, and otherorgans. It is a lethal genetic disease associated with a defect in Cltransport, and is caused by mutations in the gene coding for cysticfibrosis transmembrane conductance regulator (CFTR), a 1480 amino acidprotein that has been associated with the expression of chlorideconductance in a variety of eukaryotic cell types. Defects in CFTRdestroy or reduce the ability of epithelial cells in the airways, sweatglands, pancreas and other tissues to secret Cl in response tocAMP-mediated agonists and impair activation of apical membrane channelsby cAMP-dependent protein kinase A (PKA). Given the high incidence anddevastating nature of this disease, development of effective CFtreatments is imperative.

[0161] The CFTR gene (˜600 kb) can be transferred, such as from a CF-YAC(see Green et al. Science 250:94-98) by construction of a selectable CFYAC by inserting a selectable marker, such as puromycin or hygromycinresistance and λ-DNA by site specific integration into theneo-minichromosome or into a SATAC. The CF-YAC can be introduced intocells, such as EC3/7C5 or 19C5xHa4 by yeast protoplast fusion ormicroinjection of yeast nuclei into mammalian cells, select stabletransformants, and establish antibiotic-resistant cell lines.

[0162] 2. Disease Resistant Animals and Plants

[0163] Artificial chromosomes are ideally suited for preparing diseaseresistant animals, including vertebrates and invertebrates, includingfish as well as mammals. In particular, multivalent vaccines can beprepared. Such vaccines will be encoded by multiple antigens that can beincluded in a MAC and either delivered to a host to induce immunity, orincorporated into embryos to produce disease-resistant animals andplants (or plants and animals that are less susceptible).

[0164] Fish and crustaceans will serve as model hosts for production ofdisease resistant animals.

[0165] 3. Use of MACs and Other Artificial Chromosomes for Preparationof and Screening of Libraries

[0166] Since large fragments can incorporated into each SATAC, entiregenomes can be readily screened. For example, DNA encoding tree growthfactors can be introduced into trees. Libraries can be prepared,introduce large fragments into chromosomes, and introduce them all intotrees, thereby insuring expression.

[0167] The following examples are included for illustrative purposesonly and are not intended to limit the scope of the invention.

EXAMPLE 1

[0168] General Materials and Methods

[0169] The following materials and methods are exemplary of methods thatare used in the following Examples and that can be used to prepare celllines containing artificial chromosomes. Other suitable materials andmethods known to those of skill in the art may used. Modifications ofthese materials methods known to those of skill in the art may also beemployed.

[0170] A. Culture of Cell Lines, Cell Fusion, and Transfection of Cells

[0171] 1. Chinese hamster K20 cells and mouse A9 fibroblast cells werecultured in F-12 medium. EC3/7 (see, U.S. Pat. No. 5,288,625, anddeposited at the European Collection of Animal cell Culture (ECACC)under accession no. 90051001; see, also Hadlaczky et al. (1991) Proc.Natl. Acad. Sci. U.S.A. 88:8106-8110 and U.S. application Ser. No.08/375,271) and EC3/7C5 (see, U.S. Pat. No. 5,288,625 and Praznovszky etal. (1991) Proc. Natl. Acad. Sci. U.S.A. 88:11042-11046) mouse celllines, and the KE1-2/4 hybrid cell line were maintained in F-12 mediumcontaining 400 μg/ml G-418 (SIGMA, St. Louis, Mo.).

[0172] 2. TF1004G19 and TF1004G19C5 mouse cells, described below, andthe 19xHa4 hybrid, described below, and its sublines were cultured inF-12 medium containing 400 μg/ml Hygromycin B (Calbiochem). LP11 cellswere maintained in F-12 medium containing 3-15 μg/ml Puromycin (SIGMA,St. Louis, Mo.).

[0173] 3. Cotransfection of EC3/7C5 cells with plasmids (pH132, pCH110available from Pharmacia, see, also Hall et al. (1983) J. Mol. Appl.Gen. 2:101-109) and with λ DNA was made using the calcium phosphate DNAprecipitation method (see, e.g., Chen et al. (1987) Mol. Cell. Biol.7:2745-2752), using 2-5 μg plasmid DNA and 20 μg λ phage DNA per 5×10⁶recipient cells.

[0174] 4. Cell Fusion

[0175] Mouse and hamster cells were fused using polyethylene glycol(Davidson et al. (1976) Som. Cell Genet. 2:165-176). Hybrid cells wereselected in HAT medium containing 400 μg/ml Hygromycin B.

[0176] Approximately 2×10⁷ recipient and 2×10⁶ donor cells were fusedusing polyethylene glycol (Davidson et al. (1976) Som. Cell Genet.2:165-176). Hybrids were selected and maintained in F-12/HAT medium(Szybalsky et al. (1962) Natl. Cancer Inst. Monogr. 7:75-89) containing10% FCS and 400 μg/ml G418. The presence of “parental” chromosomes inthe hybrids cell lines was verified by in situ hybridizations withspecies-specific probes using biotin labeled human and hamster genomicDNA, and a mouse long interspersed repetitive DNA (pMCPE1.51).

[0177] 5. Microcell Fusion

[0178] Microcell-mediated chromosome transfer was done according toSaxon et al. ((1985) Mol. Cell. Biol. 1:140-146) with the modificationsof Goodfellow et al. ((1989) Techniques for mammalian genome transfer.In Genome Analysis a Practical Approach. K. E. Davies, ed., IRL Press,Oxford, Washington D.C. pp. 1-17) and Yamada et al. ((1990) Oncogene5:1141-1147). Briefly, 5×10⁶ EC3/7C5 cells in a T25 flask were treatedfirst with 0.05 μg/ml colcemid for 48 hr and then with 10 μg/mlcytochalasin B for 30 min. The T25 flasks were centrifuged on edge andthe pelleted microcells were suspended in serum free DME medium. Themicrocells were filtered through first a 5 micron and then a 3 micronpolycarbonate filter, treated with 50 μg/ml of phytohemagglutin, andused for polyethylene glycol mediated fusion with recipient cells.Selection of cells containing the MMCneo was started 48 hours afterfusion in medium containing 400-800 μg/ml G418.6

[0179] B. Chromosome Banding

[0180] Trypsin G-banding of chromosomes was performed using the methodof Wang & Fedoroff ((1972) Nature 235:52-54), and the detection ofconstitutive heterochromatin with the BSG. C-banding method was doneaccording to Sumner ((1972) Cell Res. 75:304-306). For the detection ofchromosome replication by bromodeoxyuridine (BrdU) incorporation, theFluorescein Plus Giemsa (FPG) staining method of Perry & Wolff ((1974)Nature 251:156-158) was used.

[0181] C. Immunolabelling of Chromosomes and In Situ Hybridization

[0182] Indirect immunofluorescence labelling with human anti-centromereserum LU851 (Hadlaczky et al. (1986) Exp. Cell Res. 167:1-15, andindirect immunofluorescence and in situ hybridization on the samepreparation were performed as described previously (see, Hadlaczky etal. (1991) Proc. Natl. Acad. Sci. U.S.A. 88:8106-8110, see, also U.S.application Ser. No. 08/375,271). Immunolabelling withfluorescein-conjugated anti-BrdU monoclonal antibody (Boehringer) wasperformed according to the procedure recommended by the manufacturer,except that for treatment of mouse A9 chromosomes, 2 M hydrochloric acidwas used at 37° C. for 25 min, and for chromosomes of hybrid cells, 1 Mhydrochloric acid was used at 37° C. for 30 min.

[0183] D. Scanning Electron Microscopy

[0184] Preparation of mitotic chromosomes for scanning electronmicroscopy using osmium impregnation was performed as describedpreviously (Sumner (1991) Chromosoma 100:410-418). The chromosomes wereobserved with a Hitachi S-800 field emission scanning electronmicroscope operated with an accelerating voltage of 25 kV.

[0185] E. DNA Manipulations, Plasmids and Probes

[0186] 1. General Methods

[0187] All general DNA manipulations were performed by standardprocedures (see, e.g., Sambrook et al. (1989) Molecular cloning: ALaboratory Manual Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.). The mouse major satellite probe was provided by Dr. J. B.Rattner (University of Calgary, Alberta, Canada). Cloned mouse satelliteDNA probes (see, Wong et al. (1988) Nucl. Acids Res. 16:11645-11661),including the mouse major satellite probe, were gifts from Dr. J. B.Rattner, University of Calgary. Hamster chromosome painting was donewith total hamster genomic DNA, and a cloned repetitive sequencespecific to the centromeric region of the chromosome 2 (Fátyol et al.(1994) Nucl. Acids Res. 22:3728-3736) was also used. Mouse chromosomepainting was done with a cloned long interspersed repetitive sequence(pMCP1.51) specific for the mouse euchromatin.

[0188] For cotransfection and for in situ hybridization, the pCH110β-galactosidase construct (Pharmacia or Invitrogen), and λcl 875 Sam7phage DNA (New England Biolabs) were used.

[0189] 2. Construction of Plasmid pPuroTel

[0190] Plasmid pPuroTel, which carries a Puromycin resistance gene and acloned 2.5 kb human telomeric sequence (see, SEQ ID No 3), wasconstructed from the pBabe-puro retroviral vector (Morgenstern et al.(1990) Nucl. Acids Res. 18:3587-3596; provided by Dr. L. Székely(Microbiology and Tumorbiology Center, Karolinska Institutet,Stockholm); see, also Tonghua et al. (1995) Chin. Med. J. (Beijing,Engl. Ed.) 108:653-659; Couto et al. (1994) Infect. Immun. 62:2375-2378;Dunckley et al. (1992) FEBS Lett. 296:128-34; French et al. (1995) Anal.Biochem. 228:354-355; Liu et al. (1995) Blood 85:1095-1103;International PCT application Nos. WO 9520044; WO 9500178, and WO9419456).

[0191] F. Deposited Cell Lines

[0192] Cell lines KE1 2/4, EC3/7C5, TF1004G19C5, 19C5xHa4, G3D5 and H1D3and have been deposited in accord with the Budapest Treaty at theEuropean Collection of Animal cell Culture (ECACC) under Accession Nos.96040924, 96040925, 96040926, 96040927, 96040928 and 96040929,respectively.

EXAMPLE 2

[0193] Preparation EC3/7, EC3/7C5 and Related Cell Lines

[0194] The EC3/7 cell line is an LMTK⁻ mouse cell line that contains theneo-centromere. The EC3/7C5 cell line is a single-cell subclone of EC3/7that contains the neo-minichromosome.

[0195] A. EC3/7 Cell Line

[0196] As described in U.S. Pat. No. 5,288,625 (see, also Praznovszky etal. (1991) Proc. Natl. Acad. Sci. U.S.A. 88:11042-11046 and Hadlaczky etal. (1991) Proc. Natl. Acad. Sci. U.S.A. 88:8106-8110) de novocentromere formation occurs in a transformed mouse LMTK-fibroblast cellline (EC3/7) after cointegration of A constructs (λCM8 and λgtWESneo)carrying human and bacterial DNA.

[0197] By cotransfection of a 14 kb human DNA fragment cloned in lambda(λCM8) and a dominant marker gene (λgtWESneo), a selectable centromerelinked to a dominant marker gene (neo-centromere) was formed in mouseLMTK⁻ cell line EC3/7 (Hadlaczky et al. (1991) Proc. Natl. Acad. Sci.U.S.A. 88:8106-8110, see FIG. 1) Integration of the heterologous DNA(the λ DNA and marker gene-encoding DNA) occurred into the short arm ofan acrocentric chromosome (chromosome 7 (see, FIG. 1B)), where anamplification process resulted in the formation of the new centromere(neo-centromere (see FIG. 1C)). On the dicentric chromosome (FIG. 1C)the newly formed centromere region contains all the heterologous DNA(human, lambda, and neo) introduced into the cell, and an activecentromere.

[0198] Having two functionally active centromeres on the same chromosomecauses regular breakages between the centromeres (see, FIG. 1E). Thedistance between the two centromeres on the dicentric chromosome isestimated to be ˜10-15 Mb, and the breakage that separates theminichromosome occurred between the two centromeres. Such specificchromosome breakages result in the appearance (in approximately 10% ofthe cells) of a chromosome fragment that carries the neo-centromere(FIG. 1F). This chromosome fragment is principally composed of human,lambda, plasmid, and neo gene DNA, but it also has some mousechromosomal DNA. Cytological evidence suggests that during thestabilization of the MMCneo, there was an inverted duplication of thechromosome fragment bearing the neo-centromere. The size ofminichromosomes in both cell lines is approximately 20-30 Mb; thisfinding indicates a two-fold increase in size.

[0199] From the EC3/7 cell line, which contains the dicentric chromosome(FIG. 1E), two sublines (EC3/7C5 and EC3/7C6) were selected by repeatedsingle-cell cloning. In these cell lines, the neo-centromere was foundexclusively on a small chromosome (neo-minichromosome), while theformerly dicentric chromosome carried detectable amounts of theexogenously-derived DNA sequences but not an active neo-centromere(FIGS. 1F and 1G).

[0200] The minichromosomes of cell lines EC3/7C5 and EC3/7C6 aresimilar. No differences are detected in their architectures at eitherthe cytological or molecular level. The minichromosomes wereindistinguishable by conventional restriction endonuclease mapping or bylong-range mapping using pulsed field electrophoresis and Southernhybridization. The cytoskeleton of cells of the EC3/7C6 line showed anincreased sensitivity to colchicine, so the EC3/7C5 line was used forfurther detailed analysis.

[0201] B. Preparation of the EC3/7C5 and EC3/7C6 Cell Lines

[0202] Integration of the “foreign” DNA, and subsequent amplification ofmouse and “foreign” DNA that leads to the formation of a dicentricchromosome, occurs at the centromeric region of the short arm of a mousechromosome, identified by G-banding as mouse chromosome 7. Because ofthe presence of two functionally active centromeres on the samechromosome, regular breakages occur between the centromeres. Suchspecific chromosome breakages give rise to the appearance (inapproximately 10% of the cells) of a chromosome fragment carrying theneo-centromere and the remaining portion of he formerly dicentricchromosome. From the EC3/7 cell line carrying either the dicentricchromosome or a chromosome fragment with the neo-centromere, twosublines (EC3/7C5 and EC3/7C6) were selected by repeated single-cellcloning. In these cell lines, the neo-centromere was found exclusivelyon a minichromosome (neo-minichromosome), while the formerly dicentricchromosome carried traces of “foreign” DNA sequences.

[0203] The EC3/7C5 cells, which contain the dicentric chromosome, wereproduced by subcloning the EC3/7 cell line in high concentrations ofG418 (40-fold the lethal dose) for 350 generations. Two singlecell-derived stable cell lines (EC3/7C5 and EC3/7C6) were established.These cell lines carry the neo-centromere on minichromosomes and alsocontain the remaining fragment of the dicentric chromosome. Indirectimmunofluorescence with anti-centromere antibodies and subsequent insitu hybridization experiments demonstrated that the minichromosomesderived from the dicentric chromosome. In EC3/7C5 and EC3/7C6 cell lines(140 and 128 metaphases, respectively) no dicentric chromosomes werefound and minichromosomes were detected in 97.2% and 98.1% of the cells,respectively. The minichromosomes have been maintained for over 150 cellgenerations. They do contain the remaining portion of the formerlydicentric chromosome.

[0204] Multiple copies of telomeric DNA sequences were detected in themarker centromeric region of the dicentric chromosome by in situhybridization. This indicates that mouse telomeric sequences werecoamplified with the foreign DNA sequences. These stableminichromosome-carrying cell lines provide direct evidence that theextra centromere containing human DNA is functioning and is capable ofmaintaining the minichromosomes (see, U.S. Pat. No. 5,288,625).

[0205] The chromosome breakage in the EC3/7 cells, which separates theneo-centromere from the mouse chromosome, occurred in the G-bandpositive “foreign” DNA region. This is supported by the observation oftraces of λ and human DNA sequences at the broken end of the formerlydicentric chromosome. Comparing the G-band pattern of the chromosomefragment carrying the neo-centromere with that of the stableneo-minichromosome, reveals that the neo-minichromosome is an invertedduplicate of the chromosome fragment that bears the neo-centromere. Thisis also evidenced by the observation that although theneo-minichromosome carries only one functional centromere, both ends ofthe minichromosome are heterochromatic, and mouse satellite DNAsequences were found in these heterochromatic regions by in situhybridization.

[0206] These two cell lines EC3/7C5 and EC3/7C6, thus, carry aselectable mammalian minichromosome (MMCneo) with a centromere linked toa dominant marker gene Hadlaczky et al. (1991) Proc. Natl. Acad. Sci.U.S.A. 88:8106-8110). MMCneo is intended to be used as a vector forminichromosome-mediated gene transfer and has been used as model of aminichromosome-based vector system.

[0207] Long range mapping studies of the MMCneo indicated that human DNAand the neo gene constructs integrated into the mouse chromosomeseparately, followed by the amplification of the chromosome region thatcontains the exogenous DNA. The MMCneo contains about 30-50 copies ofthe λCM8 and λgtWESneo DNA in the form of approximately 160 kb repeatedblocks, which together cover at least a 3.5 Mb region. In addition tothese, there are mouse telomeric sequences (Praznovszky et al. (1991)Proc. Natl. Acad. Sci. U.S.A. 88:11042-11046) and any DNA of mouseorigin necessary for the correct higher-ordered structural organizationof chromatids.

[0208] Using a chromosome painting probe mCPE1.51 (mouse longinterspersed repeated DNA), which recognizes exclusively euchromaticmouse DNA, detectable amounts of interspersed repeat sequences werefound on the MMCneo by in situ hybridization. The neo-centromere isassociated with a small but detectable amount of satellite DNA. Thechromosome breakage that separates the neo-centromere from the mousechromosome occurs in the “foreign” DNA region. This is demonstrated bythe presence of lambda and human DNA at the broken end of the formerlydicentric chromosome. At both ends of the MMCneo, however, there tracesof mouse major satellite DNA occur as evidence by in situ hybridization.This observation suggests that the doubling in size of the chromosomefragment carrying the neo-centromere during the stabilization of theMMCneo is a result of an inverted duplication. Although mouse telomeresequences, which coamplified with the exogenous DNA sequences during theneo-centromere formation, may provide sufficient telomeres for theMMCneo, the duplication could have supplied the functional telomeres forthe minichromosome.

[0209] C. Isolation and Partial Purification of Minichromosomes

[0210] Mitotic chromosomes of EC3/7C5 cells were isolated as describedby Hadlaczky et al. ((1981) Chromosoma 81:537-555), using aglycine-hexylene glycol buffer system (Hadlaczky et al. (1982)Chromosoma 86:643-659). Chromosome suspensions were centrifuged at1,200×g for 30 minutes. The supernatant containing minichromosomes wascentrifuged at 5,000×g for 30 minutes and the pellet was resuspended inthe appropriate buffer. Partially purified minichromosomes were storedin 50% glycerol at −20° C.

[0211] D. Stability of the MMCneo Maintenance and Neo Expression

[0212] EC3/7C5 cells grown in non-selective medium for 284 days and thentransferred to selective medium containing 400 μg/ml G418 showed a 96%plating efficiency (colony formation) compared to control cells culturedpermanently in the presence of G418. Cytogenetic analysis indicated thatthe MMCneo is stably maintained at one copy per cell both underselective and non-selective culture conditions. Only two metaphases withtwo MMCneo were found in 2,270 metaphases analyzed.

[0213] Southern hybridization analysis showed no detectable changes inDNA restriction patterns, and similar hybridization intensities wereobserved with a neo probe when DNA from cells grown under selective ornon-selective culture conditions were compared.

[0214] Northern analysis of RNA transcripts from the neo gene isolatedfrom cells grown under selective and non-selective conditions showedonly minor and not significant differences. Expression of the neo genepersisted in EC3/7C5 cells maintained in F-12 medium free of G418 for290 days under non-selective culture conditions. The long termexpression of the neo gene(s) from the minichromosome may be influencedby the nuclear location of the MMCneo. In situ hybridization experimentsrevealed a preferential peripheral location of the MMCneo in theinterphase nucleus. In more than 60% of the 2,500 nuclei analysis, theminichromosome was observed at the perimeter of the nucleus near thenuclear envelope.

EXAMPLE 3

[0215] Minichromosome Transfer and Production of the λ-Neo-Chromosome

[0216] A. Minichromosome Transfer

[0217] The neo-minichromosome (referred MMCneo, FIG. 2C) has been usedfor gene transfer by fusion of minichromosome containing cells (EC3/7C5or EC3/7C6) with different mammalian cells, including hamster and human.Thirty-seven stable hybrid cell lines have been produced. Allestablished hybrid cell lines proved to be true hybrids as evidenced byin situ hybridization using biotinylated human, and hamster genomic, orpMCPE1.51 mouse long interspersed repeated DNA probes for “chromosomepainting”.

[0218] The MMCneo has also been successfully transferred into mouse A9,L929 and pluripotent F9 teratocarcinoma cells by fusion of microcellsderived from EC3/7C5 cells. Transfer was confirmed by PCR, Southernblotting and in situ hybridization with minichromosome-specific probes.The cytogenetic analysis confirmed that, as expected for microcellfusion, a few cells (1-5%) received (or retained) the MMCneo.

[0219] These demonstrate that the MMCneo is tolerated by a wide range ofcells. The prokaryotic genes and the extra dosage for the human andlambda sequences carried on the minichromosome seem to be notdisadvantageous for tissue culture cells.

[0220] The MMCneo is the smallest chromosome of the EC3/7C5 genome andis estimated to be approximately 20-30 Mb, which is significantlysmaller than the majority of the host cell (mouse) chromosomes. Byvirtue of the smaller size, minichromosomes can be partially purifiedfrom a suspension of isolated chromosomes by a simple differentialcentrifugation. In this way, minichromosome suspensions of 15-20% purityhave been prepared. These enriched minichromosome preparations can beused to introduce, such as by microinjection or lipofection theminichromosome into selected target cells. Target cells includetherapeutic cells that can be use in methods of gene therapy, and alsoembryonic cells for the preparation of transgenic animals.

[0221] The MMCneo is capable of autonomous replication, is stablymaintained in cells, and permits persistent expression of the neogene(s), even after long-term culturing under non-selective conditions.It is a non-integrative vector that appears to occupy a territory nearthe nuclear envelope. Its peripheral localization in the nucleus mayhave an important role in maintaining the functional integrity andstability of the MMCneo. Functional compartmentalization of the hostnucleus may have an effect on the function of foreign sequences. Inaddition, contains megabases of lambda DNA sequences that should serveas a target site for homologous recombination and thus integration ofdesired gene(s) into the MMCneo. It can be transferred by cell andmicrocell fusion, microinjection, electroporation, or chromosome uptake.The neo-centromere of the MMCneo is capable of maintaining andsupporting the normal segregation of a larger 150-200 Mb λneochromosome. This result (B) demonstrates that the MMCneo chromosomeshould be useful for carrying large fragments of heterologous DNA.

[0222] B. Production of the λ-Neo-Chromosome

[0223] In one hybrid cell line KE1-2/4 made by fusion of EC3/7 andChinese hamster ovary cells (FIG. 2D), the separation of theneo-centromere from the dicentric chromosome was associated with afurther amplification process. This amplification resulted in theformation of a stable, chromosome of average size (i.e., the λneo-chromosome; see, Praznovszky et al. (1991) Proc. Natl. Acad. Sci.U.S.A. 88:11042-11046). The λ neo-chromosome carries a terminallylocated functional centromere, and is composed of seven large ampliconscontaining multiple copies of λ, human, bacterial, and mouse DNAsequences (see FIG. 2). The amplicons are separated by mouse majorsatellite DNA (Praznovszky et al. (1991) Proc. Natl. Acad. Sci. U.S.A.88:11042-11046) which forms narrow bands of constitutive heterochromatinbetween the amplicons.

EXAMPLE 4

[0224] Formation of the “Sausage Chromosome” (SC)

[0225] The findings set forth in the above EXAMPLES demonstrate that thecentromeric region of the mouse chromosome 7 has the capacity forlarge-scale amplification (other results indicate that this capacity isnot unique to chromosome 7). This conclusion is further supported byresults from cotransfection experiments, in which a second dominantselectable marker gene and a non-selected marker gene were introducedinto EC3/7 cells carrying the formerly dicentric chromosome 7 and theneo-minichromosome. The EC3/7C5 cell line was transformed with λ phageDNA, a hygromycin construct (pH132), and a β-galactosidase construct(pCH110). Stable transformants were selected in the presence of highconcentrations (400 μg/ml) Hygromycin B, and analyzed by Southernhybridization. Established transformant cell lines showing multiplecopies of integrated exogenous DNA were studied by in situ hybridizationto localize the integration site(s), and by LacZ staining to detectβ-galactosidase expression.

[0226] A. Materials and Methods

[0227] 1. Construction of pH132

[0228] The pH132 plasmid carries the hygromycin B resistance gene andthe anti-HIV-1 gag ribozyme (see, SEQ ID NO. 6 for DNA sequence thatcorresponds to the sequence of the ribozyme) under control of theβ-actin promoter. This plasmid was constructed from pHyg plasmid (Sugdenet al. (1985) Mol. Cell. Biol. 5:410-413; a gift from Dr. A. D. Riggs,Beckman Research Institute, Duarte; see, also, e.g., U.S. Pat. No.4,997,764), and from pPC-RAG12 plasmid (see, Chang et al. (1990) ClinBiotech 2:23-31; provided by Dr. J. J. Rossi, Beckman ResearchInstitute, Duarte; see, also U.S. Pat. Nos. 5,272,262, 5,149,796 and5,144,019, which describes the anti-HIV gag ribozyme and construction ofa mammalian expression vector containing the ribozyme insert linked tothe β-actin promoter and SV40 late gene transcriptional termination andpolyA signals). The ribozyme insert flanked by BamHI linkers wasinserted into BamHI-digested pHβ-Apr-1gpt (see, Gunning et al. (1987)Proc. Natl. Acad. Sci. U.S.A. 84:4831-4835, see, also U.S. Pat. No.5,144,019). An EcoRI/XhoI fragment of this vector was inserted intoEcoRI/XhoI-digested pHyg.

[0229] Plasmid pH132 was constructed as follows. First, pPC-RAG12(described by Chang et al. (1990) Clin. Biotech. 2:23-31) was digestedwith BamHI to excise a fragment containing an anti-HIV ribozyme gene(referred to as ribozyme D by Chang et al. ((1990) Clin. Biotech.2:23-31); see also U.S. Pat. No. 5,144,019 to Rossi et al., particularlyFIG. 4 of the patent) flanked by the human β-actin promoter at the 5′end of the gene and the SV40 late transcriptional termination andpolyadenylation signals at the 3′ end of the gene. As described by Changet al. ((1990) Clin. Biotech. 2:23-31), ribozyme D is targeted forcleavage of the translational initiation region of the HIV gag gene.This fragment of pPC-RAG12 was subcloned into pBluescript-KS(+)(Stratagene, La Jolla, Calif.) to produce plasmid 132. Plasmid 132 wasthen digested with XhoI and EcoRI to yield a fragment containing theribozyme D gene flanked by the β-actin promoter at the 5′ end and theSV40 termination and polyadenylation signals at the 3′ end of the gene.This fragment was ligated to the largest fragment generated by digestionof pHyg (Sugden et al. (1985) Mol. Cell. Biol. 5:410-413) with EcoRI andSalI to yield pH132. Thus, pH132 is an ˜9.3 kb plasmid containing thefollowing elements: the β-actin promoter linked to an anti-HIV ribozymegene followed by the SV40 termination and polyadenylation signals, thethymidine kinase gene promoter linked to the hygromycin resistance genefollowed by the thymidine kinase gene polyadenylation signal, and the E.coli ColE1 origin of replication and the ampicillin-resistance gene.

[0230] The plasmid pHyg (see, e.g., U.S. Pat. Nos. 4,997,764, 4,686,186and 5,162,215), which confers resistance to hygromycin B usingtranscriptional controls from the HSV-1 tk gene, was originallyconstructed from pKan2 (Yates et al. (1984) Proc. Natl. Acad. Sci.U.S.A. 81:3806-3810) and pLG89 (see, Gritz et al. (1983) Gene25:179-188). Briefly pKan2 was digested with SmaI and BglII to removethe sequences derived from transposon Tn5. The hygromycin-resistance hphgene was inserted into the digested pKan2 using blunt-end ligation atthe SnaI site and “sticky-end” ligation (using 1 Weiss unit of T4 DNAligase (BRL) in 20 microliter volume) at the BglII site. The SmaI andBglII sites of pKan2 were lost during ligation.

[0231] The resulting plasmid pH132, produced from introduction of theanti-HIV ribozyme construct with promoter and polyA site into pHyg,includes the anti-HIV ribozyme under control of the β-actin promoter aswell as the Hyg gene under control of the TK promoter.

[0232] 2. Chromosome Banding

[0233] Trypsin G-banding of chromosomes was performed as described inEXAMPLE 1.

[0234] 3. Cell Cultures

[0235] TF1004G19 and TF1004G19C5 mouse cells and the 19xHa4 hybrid,described below, and its sublines were cultured in F-12 mediumcontaining 400 μg/ml Hygromycin B (Calbiochem). B. Cotransfection ofEC3/7C5 to produce TF1004G-19 Cotransfection of EC3/7C5 cells withplasmids (pH132, pCH110 available from Pharmacia, see, also Hall et al.(1983) J. Mol. Appl. Gen. 2:101-109) and with λ DNA (λcl 875 Sam 7 (NewEngland Biolabs)) was made using the calcium phosphate DNA precipitationmethod (see, e.g., Chen et al. (1987) Mol. Cell. Biol. 7:2745-2752),using 2-5 μg plasmid DNA and 20 μg λ phage DNA per 5×10⁶ recipientcells.

[0236] C. Cell Lines Containing the Sausage Chromosome

[0237] One transformant TF1004G-19 was identified. It has a high copynumber of integrated pH132 and pCH110 sequences, and a high level ofβ-galactosidase expression. G-banding and in situ hybridization with ahuman probe (CM8; see, e.g., U.S. application Ser. No. 08/375,271)revealed unexpectedly that integration had occurred in the formerlydicentric chromosome 7 of the EC3/7C5 cell line. Furthermore, thischromosome carried a newly formed heterochromatic chromosome arm. Thesize of this heterochromatic arm varied between ˜150 and ˜800 Mb inindividual metaphases.

[0238] By single cell cloning from the TF1004G-19 cell line, a subcloneTF1004G-19C5 (FIG. 2D), which carries a stable chromosome 7 with a˜100-150 Mb heterochromatic arm (the sausage chromosome) was obtained.This cell line has been deposited in the ECACC under Accession No.96040926. This chromosome arm is composed of four to five satellitesegments rich in satellite DNA, and evenly spaced integratedheterologous “foreign” DNA sequences. At the end of the compactheterochromatic arm of the sausage chromosome, a less condensedeuchromatic terminal segment is regularly observed. This subclone wasused for further analyses.

[0239] D. Demonstration that the Sausage Chromosome is Derived from theFormerly Dicentric Chromosome

[0240] In situ hybridization with lambda phage and pH132 DNA on theTF1004G-19C5 cell line showed positive hybridization only on theminichromosome and on the heterochromatic arm of the “sausage”chromosome (FIG. 2D). It appears that the “sausage” chromosome (hereinalso referred to as the SC) developed from the formerly dicentricchromosome (FD) of the EC3/7C5 cell line.

[0241] To establish this, the integration sites of pCH110 and pH132plasmids was determined. This was accomplished by in situ hybridizationon these cells with biotin-labeled subfragments of the hygromycinresistance gene and the β-galactosidase gene. Both experiments resultedin narrow hybridizing bands on the heterochromatic arm of sausagechromosome. The same hybridization pattern was detected on the sausagechromosome using a mixture of biotin-labeled λ probe and pH132 plasmid,proving the cointegration of lambda phages, pH132 and pCH110 plasmids.

[0242] To examine this further, the cells were cultured in the presenceof the DNA-binding dye Hoechst 33258. Culturing of mouse cells in thepresence of this dye results in under-condensation of the pericentricheterochromatin of metaphase chromosomes, thereby permitting betterobservation of the hybridization pattern. Using this technique theheterochromatic arm of sausage chromosome of 19C5 cell showed regularunder-condensation revealing the details of the structure of “sausage”chromosome by in situ hybridization. Results of in situ hybridization onHoechst-treated TF1004G/19C5 cells with biotin-labeled subfragments ofhygromycin resistance and 8-galactosidase genes shows that these genesare localized only in the heterochromatic arm of the sausage chromosome.In addition an equal banding hybridization pattern was observed. Thispattern of repeating units (amplicons) clearly indicates that thesausage chromosome was formed by an amplification process and that thelambda phage, pH132 and pCH110 plasmid DNA sequences border theamplicons.

[0243] In another series of experiments, using fluorescence in situhybridization (FISH) was carried out with mouse major satellite DNA, themain component of the mouse pericentric heterochromatin, the resultsconfirmed that the amplicons of the sausage chromosome are primarilycomposed of satellite DNA.

[0244] E. The Sausage Chromosome has One Centromere

[0245] To determine whether mouse centromeric sequences had participatedin the amplification process forming the “sausage” chromosome andwhether or not the amplicons carry inactive centromeres, in situhybridization was carried out with mouse minor satellite DNA. Mouseminor satellite DNA is localized specifically near the centromeres ofall mouse chromosome. Positive hybridization was detected in all mousecentromeres including the sausage chromosome, which, however, onlyshowed a positive signal at the beginning of the heterochromatic arm.

[0246] Indirect immunofluorescence with human anti-centromere antibody(LU 851) that only which can recognizes the functional centromeres (see,e.g., Hadlaczky et al. (1989) Chromosoma 97:282-288) proved that sausagechromosome has only one active centromere. The centromere comes from theformerly dicentric part of the chromosome and colocalizes with the insitu hybridization signal of the mouse minor DNA probe.

[0247] F. The Selected and Non-Selected Heterologous DNA in theHeterochromatin of the Sausage Chromosome is Expressed

[0248] 1. High Levels of the Heterologous Genes are Expressed

[0249] The TF1004G/19C5 cell line thus carries multiple copies ofhygromycin resistance and β-galactosidase genes localized only in theheterochromatic arm of the sausage chromosome. The 19C5 cells can growvery well in the presence of 200 μg/ml or even 400 μg/ml hygromycin B.(The level of expression was determined by Northern hybridization withsubfragment of hygromycin resistance gene and single copy gene).

[0250] The expression of the non-selected β-galactosidase gene in theTF1004G/19C5 transformant, was detected with LacZ staining of the cells.By this method one hundred percent of the cells stained dark blue,showing that there is a high level of β-galactosidase expression in allof 19C5 cells.

[0251] 2. The Heterologous Genes that are Expressed are in theHeterochromatin

[0252] To demonstrate that the genes localized in the constitutiveheterochromatin of the sausage chromosome provide the hygromycinresistance and the LacZ staining capability of 19C5 transformant (i.e.β-gal express), PEG induced cell fusion between a TF1004G/19C5 mousecell and Chinese hamster ovary cell was performed. The hybrids wereselected and maintained in HAT medium containing G418 (400 μg/ml) andhygromycin (200 μg/ml). Two hybrid clones designated 19C5xHa3 and19C5xHa4, which has been deposited in the ECACC under Accession No.96040927, were selected. Both carry the sausage chromosome and theminichromosome.

[0253] Twenty-seven single cell derived colonies of 19C5xHa4 hybrid weremaintained and analyzed as individual subclones. In situ hybridizationwith hamster and mouse chromosome painting probes and hamster chromosome2 specific probes verified that the 19C5xHa4 clone contains the completeChinese hamster genome and a partial mouse genome. All 19C5xHa4subclones retained the hamster genome, but different subclones showeddifferent number of mouse chromosomes indicating the preferentialelimination of mouse chromosomes.

[0254] To promote further elimination of mouse chromosomes hybrid cellswere repeatedly treated with BrdU. The BrdU treatments, whichdestabilize the genome, result in significant loss of mouse chromosomes.The BrdU treated 19C5xHa4 hybrid cells were divided to three groups. Onegroup of the hybrid cells (GH) was maintained in the presence ofhygromycin (200 μg/ml) and G418 (400 μg/ml), and the other two groups ofthe cells were cultured under G418 (G) or hygromycin (H) selectionconditions to promote the elimination of sausage or minichromosome.

[0255] One month later, single cell derived subclones were establishedfrom these three subcultures of the TF1004G/19C5xHa4 hybrid line. Thesubclones were monitored by in situ hybridization with biotin-labeledlambda phage and hamster chromosome painting probes. Four individualclones (G2B5, G3C5, G4D6, G2B4) selected in the presence of G418 thathave lost the sausage chromosome but retained minichromosome were found.Under hygromycin selection only one subclone (H1D3) lost theminichromosome. In this clone the sausage chromosome was present.

[0256] Since hygromycin resistance and β-galactosidase genes werethought to be expressed from the sausage chromosome, the expression ofthese genes were analyzed in the four subclones that had lost thesausage chromosome. In the presence of 200 μg/ml hygromycin, one hundredpercent of the cells of four individual subclones died. In order todetect the β-galactosidase expression hybrid subclones were analyzed byLacZ staining. One hundred percent of the cells of the four subclonesthat lost the sausage chromosome also lost the LacZ staining capability.All of the other hybrid subclones that not lost the sausage chromosomeunder the non-selective culture conditions showed positive LacZstaining.

[0257] These findings demonstrate that the expression of hygromycinresistance and β-galactosidase genes is linked to the presence of thesausage chromosome and results of in situ hybridizations show that theheterologous DNA is expressed from the constitutive heterochromatin ofthe sausage chromosome.

[0258] By in situ hybridization in three other hybrid subclones (G2C6,G2D1, and G4D5) the presence of the sausage chromosome was not detected.By the LacZ staining method some stained cells were detected in thesehybrid lines and when these subclones were transferred to hygromycinselection some colonies survived. Cytological analysis and in situhybridization of these hygromycin resistance colonies revealed thepresence of the sausage chromosome, suggesting that only the cells ofG2C6, G2D1 and G4D5 hybrids that had not lost the sausage chromosomewere able to preserve the hygromycin resistance and β-galactosidaseexpression. These results confirmed that the expression of these genesis linked to the presence of the sausage chromosome. The level ofβ-galactosidase expression was determined by the immunoblot techniqueusing monoclonal antibody.

[0259] Hygromycin resistance and β-galactosidase expression of the cellswhich contained the sausage chromosome were provided by the geneslocalized in the mouse pericentric heterochromatin. This wasdemonstrated by performing Southern DNA hybridizations on the hybridcells that lack the sausage chromosome with PCR amplified subfragmentsof hygromycin resistance and β-galactosidase genes. None of thesubclones showed hybridization with these probes, however, all of theanalyzed clones contained the minichromosome as well. Other hybridclones that contain the sausage chromosome showed intense hybridizationwith these DNA probes. These results lead to the conclusion thathygromycin resistance and β-galactosidase expression of the cells thatcontain the sausage chromosome were provided by the genes localized inthe mouse pericentric heterochromatin.

EXAMPLE 5

[0260] The Gigachromosome

[0261] As described in Example 4, the sausage chromosome was transferredinto Chinese hamster cells by cell fusion. Using Hygromycin B/HATselection, two hybrid clones 19C5xHa3 and 19C5xHa4 were produced thatcarry sausage chromosome. In situ hybridization, using hamster and mousechromosome-painting probes and a hamster chromosome 2 specific probe,verified that clone 19C5xHa4 contains a complete Chinese hamster genomeas well as partial mouse genomes. Twenty-seven separate colonies of19C5xHa4 cells were maintained and analyzed as individual subclones.Twenty-six out of 27 subclones contained a morphologically unchangedsausage chromosome.

[0262] In one clone 19C5xHa47 (see FIG. 2E), the heterochromatic arm ofthe sausage chromosome became unstable and showed continuousintrachromosomal growth. In extreme cases, the amplified chromosome armexceeded 1000 Mb in size (gigachromosome).

EXAMPLE 6

[0263] The Stable Megachromosome

[0264] A. Formation of the Megachromosome

[0265] All 19C5xHa4 subclones retained a complete hamster genome, butdifferent subclones showed different numbers of mouse chromosomes,indicating the preferential elimination of mouse chromosomes. Asdescribed in Example 4, to promote further elimination of mousechromosomes, hybrid cells were repeatedly treated with 10⁻⁴ M BrdU for16 hours and single cell subclones were established. The BrdU treatmentsappeared to destabilize the genome, resulting in a change in the sausagechromosome as well. A gradual increase in a cell population in which afurther amplification had occurred was observed. In addition to the˜100-150 Mb heterochromatic arm of the sausage chromosome, an extracentromere and a ˜150-250 Mb heterochromatic chromosome arm were formed,which differed from those of mouse chromosome 7. By the acquisition ofanother euchromatic terminal segment, a new submetacentric chromosome(megachromosome) was formed. Seventy-nine individual subclones wereestablished from these BrdU-treated cultures, by single-cell cloning: 42subclones carried the intact megachromosome, 5 subclones carried thesausage chromosome, and in 32 subclones fragments or translocatedsegments of the megachromosome were observed. Twenty-six subclones werecultured under non-selective conditions over a 2 month period. In 19 outof 26 subclones, the megachromosome was retained. Those subclones whichlost the megachromosomes all became sensitive to Hygromycin B and had noβ-galactosidase expression, indicating that both markers were linked tothe megachromosome.

[0266] Two sublines (G3D5 and H1D3), which were chosen for furtherexperiments, showed no changes in the morphology of the megachromosomeduring more than 100 generations under selective conditions.

[0267] B. Structure of the Megachromosome

[0268] The following results demonstrate that, apart from theeuchromatic terminal segments, the whole megachromosome is constitutiveheterochromatin, containing a tandem array of at least 40 (˜7.5 Mb)blocks of mouse major satellite DNA (see FIGS. 2 and 3). Four satelliteDNA blocks are organized into a giant palindrome (amplicon) carryingintegrated exogenous DNA sequences at each end. The long and short armsof the submetacentric megachromosome contains 6 and 4 amplicons,respectively.

[0269] 1. The Megachromosome is Composed Primarily of Heterochromatin

[0270] Except for the terminal regions, the megachromosome is composedprimarily of heterochromatin. This was demonstrated by G-banding of themegachromosome, which resulted in positive staining characteristic ofconstitutive heterochromatin. Apart from the terminal regions, the wholemegachromosome appears to be heterochromatic. Mouse major satellite DNAis the main component of the pericentric, constitutive heterochromatinof mouse chromosomes and represents ˜10% of the total DNA (Waring et al.(1966) Science 154:791-794). Using a mouse major satellite DNA probe forin situ hybridization, strong hybridization was observed throughout themegachromosome, except for its terminal regions. The hybridizationshowed a segmented pattern: four large blocks appeared on the short armand usually 4-7 blocks were seen on the long arm. By comparing thesesegments with the pericentric regions of normal mouse chromosomes thatcarry approximately ˜15 Mb of major satellite, the size of the blocks ofmajor satellite on the megachromosome was estimated to be ˜30 Mb.

[0271] Using a mouse probe specific to euchromatin (pMCPE1.51; a mouselong interspersed repeated DNA probe), positive hybridization wasdetected only on the terminal segments of the megachromosome of the H1D3hybrid subline. In the G3D5 hybrids, hybridization with ahamster-specific probe revealed that several megachromosomes containedterminal segments of hamster origin on the long arm. This observationindicated that the acquisition of the terminal segments on thesechromosomes happened in the hybrid cells, and that the long arm of themegachromosome was the recently formed one. When a mouse minor satelliteprobe was used, specific to the centromeres of mouse chromosomes (Wonget al. (1988) Nucl. Acids Res. 16:11645-11661), a strong hybridizationsignal was detected only at the primary constriction of themegachromosome, which colocalized with the positive immunofluorescencesignal produced with human anti-centromere serum (LU 851).

[0272] In situ hybridization experiments with pH132, pCH110, and λ DNAprobes revealed that all heterologous DNA was located in the gapsbetween the mouse major satellite segments. Each segment of mouse majorsatellite was bordered by a narrow band of integrated heterologous DNA,except at the second segment of the long arm where a double bandexisted, indicating that the major satellite segment was missing orconsiderably reduced in size here. This chromosome region served as auseful cytological marker in identifying the long arm of themegachromosome. At a frequency of 10⁻⁴, “restoration” of these missingsatellite DNA blocks was observed in one chromatid, when the formationof a whole segment on one chromatid occurred.

[0273] After Hoechst 33258 treatment (50 μg/ml for 16 hours), themegachromosome showed undercondensation throughout its length except forthe terminal segments. This made it possible to study the architectureof the megachromosome at higher resolution. In situ hybridization withthe mouse major satellite probe on undercondensed megachromosomesdemonstrated that the ˜30 Mb major satellite segments were composed offour blocks of ˜7.5 Mb separated from each other by a narrow band ofnon-hybridizing sequences (FIG. 3). Similar segmentation can be observedin the large block of pericentric heterochromatin in metacentric mousechromosomes from the LMTK⁻ and A9 cell lines.

[0274] 2. The Megachromosome is Composed of Segments Containing TwoTandem ˜7.5 Mb Blocks are Followed by Two Inverted Blocks

[0275] Because of the asymmetry in thymidine content between the twostrands of the DNA of the mouse major satellite, when mouse cells aregrown in the presence of BrdU for a single S phase, the constitutiveheterochromatin shows lateral asymmetry after FPG staining. Also, in the19C5xHa4 hybrids, the thymidine-kinase the (Tk) deficiency of the mousefibroblast cells was complemented by the hamster Tk gene, permittingBrdU incorporation experiments.

[0276] A striking structural regularity in the megachromosome wasdetected using the FPG technique. In both chromatids, alternating darkand light staining that produced a checkered appearance of themegachromosome was observed. A similar picture was obtained by labellingwith fluorescein-conjugated anti-BrdU antibody. Comparing these picturesto the segmented appearance of the megachromosome, showed that one darkand one light FPG band corresponded to one ˜30 Mb segment of themegachromosome. These results suggest that the two halves of the ˜30 Mbsegment have an inverted orientation. This was verified by combining insitu hybridization and immunolabelling of the incorporated BrdU withfluorescein-conjugated anti-BrdU antibody on the same chromosome. Sincethe ˜30 Mb segments of the megachromosome are composed of four blocks ofmouse major satellite, it can be concluded that two tandem ˜7.5 Mbblocks are followed by two inverted blocks within one segment.

[0277] Large-scale mapping of megachromosome DNA by pulsed-fieldelectrophoresis and Southern hybridization with “foreign” DNA probesrevealed a simple pattern of restriction fragments. Using endonucleaseswith none, or only a single cleavage site in the integrated foreign DNAsequences, followed by hybridization with a hyg probe, 1-4 predominantfragments were detected. Since the megachromosome contains 10-12amplicons with an estimated 3-8 copies of hyg sequences per amplicon(30-90 copies per megachromosome), the small number of hybridizingfragments indicates the homogeneity of DNA in the amplified segments.

[0278] 3. Scanning Electron Microscopy of the Megachromosome Confirmedthe Above Findings

[0279] The homogeneous architecture of the heterochromatic arms of themegachromosome was confirmed by high resolution scanning electronmicroscopy. Extended arms of megachromosomes, and the pericentricheterochromatic region of mouse chromosomes, treated with Hoechst 33258,showed similar structure. The constitutive heterochromatic regionsappeared more compact than the euchromatic segments. Apart from theterminal regions, both arms of the megachromosome were completelyextended, and showed faint grooves, which should correspond to theborder of the satellite DNA blocks in the non-amplified chromosomes andin the megachromosome. Without Hoechst treatment, the grooves seemed tocorrespond to the amplicon borders on the megachromosome arms. Inaddition, centromeres showed a more compact, finely fibrous appearancethan the surrounding heterochromatin.

[0280] C. Formation of the Megachromosome

[0281]FIG. 2 schematically sets forth the events leading to theformation of the stable megachromosome: (A) A single E-typeamplification in the centromeric region of chromosome 7 generates theneo-centromere linked to the integrated foreign DNA, and forms adicentric chromosome. Multiple E-type amplification forms the λneo-chromosome, which was derived from chromosome 7 and stabilized in amouse-hamster hybrid cell line; (B) Specific breakage between thecentromeres of a dicentric chromosome 7 generates a chromosome fragmentwith the neo-centromere, and a chromosome 7 with traces of foreign DNAat the end; (C) Inverted duplication of the fragment bearing theneo-centromere results in the formation of a stable neo-minichromosome;(D) Integration of exogenous DNA into the foreign DNA region of theformerly dicentric chromosome 7 initiates H-type amplification, and theformation of a heterochromatic arm. By capturing a euchromatic terminalsegment, this new chromosome arm is stabilized in the form of the“sausage” chromosome; (E) BrdU treatment and/or drug selection appearsto induce further H-type amplification, which results in the formationof an unstable gigachromosome: (F) Repeated BrdU treatments and/or drugselection induce further H-type amplification including a centromereduplication, which leads to the formation of another heterochromaticchromosome arm. It is split off from the chromosome 7 by chromosomebreakage and acquires a terminal segment to form the stablemegachromosome.

EXAMPLE 7

[0282] Summary of some of the Cell Lines with SATACS and Minichromosomesthat have been Constructed

[0283] LMTK⁻-derived cell line, which is a mouse fibroblast cell line,was transfected with λCM8 and λgtWESneo DNA (see, EXAMPLE 2) to producetransformed cell lines. Among these cell lines was EC3/7, deposited atthe European Collection of Animal cell Culture (ECACC) under AccessionNo. 90051001 (see, U.S. Pat. No. 5,288,625; see, also Hadlaczky et al.(1991) Proc. Natl. Acad. Sci. U.S.A. 88:8106-8110 and U.S. applicationSer. No. 08/375,271). This cell line contains the dicentric chromosomewith the neo centromere. Recloning and selection produced cell linessuch as EC3/7C5, which are cell lines with the stable neo-minichromosome(see, FIG. 2C).

[0284] Fusion of EC3/7 with CHO-K20 cells and selection with G418/HATproduced hybrid cell lines, among these was KE1 2/4, which has beendeposited with the ECACC under Accession No. 96040924. KE1 2/4 is astable cell line that contains the A neo-chromosome (see, FIG. 2D; see,also U.S. Pat. No. 5,288,625), produced by E type amplifications. KE12/4 has been transfected with vectors containing lambda DNA, selectablemarkers, such as puromycin resistance, and genes of interest, such asp53, anti-HIV ribozymes. These vectors target the gene of interest intothe λ neo-chromosome by virtue of homologous recombination with theheterologous DNA in the chromosome.

[0285] The EC3/7C5 cell line has been co-transfected with pH132, pCH110and λ DNA (see, EXAMPLE 2) as well as other constructs. Various clonesand subclones have been selected. For example transformation with aconstruct that includes p53 encoding DNA, produced cells designated C5pMCT53.

[0286] As discussed above, cotransfection of EC3/7C5 cells with plasmids(pH132, pCH110 available from Pharmacia, see, also Hall et al. (1983) J.Mol. Appl. Gen. 2:101-109) and with λ DNA (λcl 875 Sam 7 (New EnglandBiolabs)) produced transformed cells. Among these is TF1004G24, whichcontains the DNA encoding the anti-HIV ribozyme in theneo-minichromosome. Recloning of TF1004G24 produced numerous cell lines.Among these the NHHL24 cell line. This cell line also has the anti-HIVribozyme in the neo-minichromosome and expresses high levels of β-gal.It has been fused with CHO-K20 cells to produce various hybrids.Recloning and selection of the TF1004G transformants produced the cellline TF1004G-19, discussed above in EXAMPLE 4, which contains theunstable sausage chromosome. Single cell cloning produced theTF1004G-19C5 (see FIG. 4) cell line, which has a stable sausagechromosome. TF1004G-19C5 has been fused with CHO cells and the hybridsgrown under selective conditions to produce the 19C5xHa4 cell line (see,EXAMPLE 4) and others. BrdU treatment of 19C5xHa4 cells and growth underselective conditions (neomycin (G) and/or hygromycin (H)) has producedhybrid cell lines with the gigachromsome (see FIG. 2E) and the G3D5 andG4D6 cell lines and others. G3D5 has the neo-minichromosome and themegachromosome, G4D6 has only the neo-minichromosome.

[0287] Recloning of G3D5 in GH medium produced numerous clones. Amongthese is H1D3 (see FIG. 4), which has the stable megachromosome.Repeated BrDU treatment and recloning has produced the HB31 cell line,which has been used for transformations with the pTEMPUD, pTEMPU andpTEMPU3 vectors (see, Example 12, below).

[0288] H1 D3 has been fused with a CD4⁺ Hela cell line that carries DNAencoding CD4 and neomycin resistance on a plasmid (see, e.g., U.S. Pat.Nos. 5,413,914, 5,409,810, 5,266,600, 5,223,263, 5,215,914 and5,144,019, which describe these Hela cells). Selection with GH hasproduced hybrids, including H1xHE41 (see FIG. 4), which carries themegachromosome and also a single human chromosome that includes theCD4neo construct. Repeated BrdU treatment and single cell cloning hasproduced cell lines with the megachromosome (cell line 1B3, see FIG. 4),cell lines, such as 1B4 and others that have a dwarf megachromosome(˜150-200 Mb) and cell lines, such as 1C3, which hasmicro-megachromosome (˜60-90 Mb). About 25% of the 1B3 cells have atruncated megachromosome (˜90-120 Mb).

EXAMPLE 8

[0289] Replication of the Megachromosome

[0290] This homogeneous architecture of the megachromosomes provides aunique opportunity to perform a detailed analysis of the replication ofthe constitutive heterochromatin.

[0291] A. Materials and Methods

[0292] 1. Culture of Cell Lines

[0293] H1D3 mouse-hamster hybrid cells carrying the megachromosome (see,EXAMPLE 4) were cultured in F-12 medium containing 10% fetal calf serum(FCS) and 400 μg/ml Hygromycin B (Calbiochem). G3D5 hybrid cells (see,Example 4) were maintained in F-12 medium containing 10% FCS, 400 μg/mlHygromycin B (Calbiochem), and 400 μg/ml G418 (SIGMA). Mouse A9fibroblast cells were cultured in F-12 medium supplemented with 10% FCS.

[0294] 2. BrdU Labelling

[0295] In typical experiments, 20-24 parallel semi-confluent cellcultures were set up in 10 cm Petri dishes. Bromodeoxyuridine (BrdU)(Fluka) was dissolved in distilled water alkalized with a drop of NaOH,to make a 10⁻² M stock solution. Aliquots of 10-50 μl of this BrdU stocksolution were added to each 10 ml culture, to give a final BrdUconcentration of 10-50 μM. The cells were cultured in the presence ofBrdU for 30 min, and then washed with warm complete medium, andincubated without BrdU until required. At this point, 5 μg/ml colchicinewas added to a sample culture every 1 or 2 h. After 1-2 h colchicinetreatment, mitotic cells were collected by “shake-off” and regularchromosome preparations were made for immunolabelling.

[0296] 3. Immunolabelling of Chromosomes and In Situ Hybridization

[0297] Immunolabelling with fluorescein-conjugated anti-BrdU monoclonalantibody (Boehringer) was done according to the manufacturer'srecommendations, except that for mouse A9 chromosomes, 2 M hydrochloricacid was used at 37° C. for 25 min, while for chromosomes of hybridcells, 1 M hydrochloric acid was used at 37° C. for 30 min. In situhybridization with biotin-labelled probes, and indirectimmunofluorescence and in situ hybridization on the same preparation,were performed as described previously (Hadlaczky et al. (1991) Proc.Natl. Acad. Sci. U.S.A. 88:8106-8110, see, also U.S. Pat. No.5,288,625).

[0298] 4. Microscopy

[0299] All observations and microphotography were made by using a VanoxAHBS (Olympus) microscope. Fujicolor 400 Super G or Fujicolor 1600 SuperHG high-speed color negatives were used for photographs.

[0300] B. Results

[0301] The replication of the megachromosome was analyzed by BrdU pulselabelling followed by immunolabelling. The basic parameters for DNAlabelling in vivo were first established. Using a 30 min pulse of 50 μMBrdU in parallel cultures, samples were taken and fixed at 5 minintervals from the beginning of the pulse, and every 15 min up to 1 hafter the removal of BrdU. Incorporated BrdU was detected byimmunolabelling with fluorescein-conjugated anti-BrdU monoclonalantibody. At the first time point (5 min) 38% of the nuclei werelabelled, and a gradual increase in the number of labelled nuclei wasobserved during incubation in the presence of BrdU, culminating in 46%in the 30 min sample, at the time of the removal of BrdU. At furthertime points (60, 75, and 90 min) no significant changes were observed,and the fraction of labelled nuclei remaining constant (44.5-46%).

[0302] These results indicate that (i) the incorporation of the BrdU isa rapid process, (ii) the 30 min pulse-time is sufficient for reliablelabelling of S-phase nuclei, and (iii) the BrdU can be effectivelyremoved from the cultures by washing.

[0303] The length of the cell cycle of the 19C5xHa4 hybrid cells wasestimated by measuring the time between the appearance of the earliestBrdU signals on the extreme late replicating chromosome segments and theappearance of the same pattern only on one of the chromatids of thechromosomes after one completed cell cycle. The length of G2 period wasdetermined by the time of the first detectable BrdU signal on prophasechromosomes and by the labelled mitoses method (Qastler et al. (1959)Exp. Cell Res. 17:420-438). The length of the S-phase was determined inthree ways: (i) on the basis of the length of cell cycle and thefraction of nuclei labelled during the 30-120 min pulse; (ii) bymeasuring the time between the very end of the replication of theextreme late replicating chromosomes and the detection of the firstsignal on the chromosomes at the beginning of S phase; (iii) by thelabelled mitoses method. In repeated experiments, the duration of thecell cycle was found to be 22-26 h, the S phase 10-14 h, and the G2phase 3.5-4.5 h.

[0304] Analyses of the replication of the megachromosome were made inparallel cultures by collecting mitotic cells at two hour intervalsfollowing two hours of colchicine treatment. In a repeat experiment, thesame analysis was performed using one hour sample intervals and one hourcolchicine treatment. Although the two procedures gave comparableresults, the two hour sample intervals were viewed as more appropriatesince approximately 30% of the cells were found to have a considerablyshorter or longer cell cycle than the average. The characteristicreplication patterns of the individual chromosomes, especially some ofthe late replicating hamster chromosomes, served as useful internalmarkers for the different stages of S-phase. To minimize the errorcaused by the different lengths of cell cycles in the differentexperiments, samples were taken and analyzed throughout the whole cellcycle until the appearance of the first signals on one chromatid at thebeginning of the second S-phase.

[0305] The sequence of replication in the megachromosome is as follows.At the very beginning of the S-phase, the replication of megachromosomestarts at the ends of the chromosomes. The first initiation ofreplication in an interstitial position can usually be detected at thecentromeric region. Soon after, but still in the first quarter of theS-phase, when the terminal region of the short arm has almost completedits replication, discrete initiation signals appear along the chromosomearms. In the second quarter of the S-phase, as replication proceeds, theBrdU-labelled zones gradually widen, and the checkered pattern of themegachromosome becomes clear (see, e.g., FIG. 2F). At the same time,pericentric regions of mouse chromosomes also show intense incorporationof BrdU. The replication of the megachromosome peaks at the end of thesecond quarter and in the third quarter of the S-phase. At the end ofthe third quarter, and at the very beginning of the last quarter of theS-phase, the megachromosome and the pericentric heterochromatin of themouse chromosomes complete their replication. By the end of S-phase onlythe very late replicating segments of mouse and hamster chromosomes arestill incorporating BrdU.

[0306] The replication of the whole genome occurs in distinct phases.The signal of incorporated BrdU increased continuously until the end ofthe first half of the S-phase, but at the beginning of the third quarterof the S-phase chromosome segments other than the heterochromaticregions hardly incorporated BrdU. In the last quarter of the S-phase,the BrdU signals increased again when the extreme late replicatingsegments showed very intense incorporation.

[0307] Similar analyses of the replication in mouse A9 cells wereperformed as controls. To increase the resolution of the immunolabellingpattern, pericentric regions of A9 chromosomes were decondensed bytreatment with Hoechst 33258. Because of the intense replication of thesurrounding euchromatic sequences, precise localization of the initialBrdU signal in the heterochromatin was normally difficult, even onundercondensed mouse chromosomes. On those chromosomes where theinitiation signal(s) were localized unambiguously, the replication ofthe pericentric heterochromatin of A9 chromosomes was similar to that ofthe megachromosome. Chromosomes of A9 cells also exhibited replicationpatterns and sequences similar to those of the mouse chromosomes in thehybrid cells. These results indicate that the replicators of themegachromosome and mouse chromosomes retained their original timing andspecificity in the hybrid cells.

[0308] By comparing the pattern of the initiation sites obtained afterBrdU incorporation with the location of the integration sites of the“foreign” DNA in a detailed analysis of the first quarter of theS-phase, an attempt was made to identify origins of replication(initiation sites) in relation to the amplicon structure of themegachromosome. The double band of integrated DNA on the long arm of themegachromosome served as a cytological marker. The results showed acolocalization of the BrdU and in situ hybridization signals found atthe cytological level, indicating that the “foreign” DNA sequences arein close proximity to the origins of replication, presumably integratedinto the non-satellite sequences between the replicator and thesatellite sequences (see, FIG. 3). In the pericentric region of severalother chromosomes, dot-like BrdU signals can also be observed that arecomparable to the initiation signals on the megachromosome. Thesesignals may represent similar initiation sites in the heterochromaticregions of normal chromosomes.

[0309] At a frequency of 10⁻⁴, “uncontrolled” amplification of theintegrated DNA sequences was observed in the megachromosome. Consistentwith the assumption (above) that “foreign” sequences are in theproximity to the replicators, this spatially restricted amplification islikely to be a consequence of uncontrolled repeated firings of thereplication origin(s) without completing the replication of the wholesegment.

[0310] C. Discussion

[0311] It has generally been thought that the constitutiveheterochromatin of the pericentric regions of chromosomes is latereplicating (see, e.g., Miller (1976) Chromosoma 55:165-170). On thecontrary, these experiments evidence that the replication of theheterochromatic blocks starts at a discrete initiation site in the firsthalf of the S-phase and continues through approximately three-quartersof S-phase. This difference can be explained in the following ways: (i)in normal chromosomes, actively replicating euchromatic sequences thatsurround the satellite DNA obscure the initiation signals, and thus theprecise localization of initiation sites is obscured; (ii) replicationof the heterochromatin can only be detected unambiguously in a periodduring the second half of the S-phase, when the bulk of theheterochromatin replicates and most other chromosomal regions havealready completed their replication, or have not yet started it. Thus,low resolution cytological techniques, such as analysis of incorporationof radioactively labelled precursors by autoradiography, only detectprominent replication signals in the heterochromatin in the second halfof S-phase, when adjacent euchromatic segments are no longerreplicating.

[0312] In the megachromosome, the primary initiation sites ofreplication colocalize with the sites where the “foreign” DNA sequencesare integrated at the amplicon borders. Similar initiation signals wereobserved at the same time in the pericentric heterochromatin of mousechromosomes that do not have “foreign” DNA, indicating that thereplication initiation sites at the borders of amplicons may reside inthe non-satellite flanking sequences of the satellite DNA blocks. Thepresence of a primary initiation site at each satellite DNA doubletimplies that this large chromosome segment is a single huge unit ofreplication (megareplicon) delimited by the primary initiation site andthe termination point at each end of the unit. Several lines of evidenceindicate that, within this higher-order replication unit, “secondary”origins and replicons contribute to the complete replication of themegareplicon:

[0313] 1. The total replication time of the heterochromatic regions ofthe megachromosome was ˜9-11 h. At the rate of movement of replicationforks, 0.5-5 kb per minute, that is typical of eukaryotic chromosomes(Kornberg et al. (1992) DNA Replication, 2nd ed., New York: W.H. Freemanand Co., p. 474), replication of a ˜15 Mb replicon would require 50-500h. Alternatively, if only a single replication origin was used, theaverage replication speed would have to be 25 kb per minute to completereplication within 10 h. By comparing the intensity of the BrdU signalson the euchromatic and the heterochromatic chromosome segments, noevidence for a 5- to 50-fold difference in their replication speed wasfound.

[0314] 2. Using short BrdU pulse labelling, a single origin ofreplication would produce a replication band that moves along thereplicon, reflecting the movement of the replication fork. In contrast,a widening of the replication zone that finally gave rise to thecheckered pattern of the megachromosome and within the replicationperiod the most intensive BrdU incorporation occurred in the second halfof the S-phase was observed. This suggests that once the megareplicatorhas been activated, it permits the activation and firing of “secondary”origins, and that the replication of the bulk of the satellite DNA takesplace from these “secondary” origins during the second half of theS-phase. This is supported by the observation that in certain stages ofthe replication of the megachromosome, the whole amplicon can apparentlybe labelled by a short BrdU pulse.

[0315] Megareplicators and secondary replication origins seem to beunder strict temporal and spatial control. The first initiation withinthe megachromosomes usually occurred at the centromere, and that shortlyafterward all the megareplicators become active. The last segment of themegachromosome to complete replication was usually the second segment ofthe long arm. Results of control experiments with mouse A9 chromosomesindicate that replication of the heterochromatin of mouse chromosomescorresponds to the replication of the megachromosome amplicons.Therefore, the pre-existing temporal control of replication in theheterochromatic blocks is preserved in the megachromosome. Positive(Hassan et al. (1994) J. Cell. Sci. 107:425-434) and negative (Haase etal. (1994) Mol. Cell. Biol. 14:2516-2524) correlations betweentranscriptional activity and initiation of replication have beenproposed. In the megachromosome, transcription of the integrated genesseems to have no effect on the original timing of the replicationorigins. The concerted, precise timing of the megareplicator initiationsin the different amplicons suggests the presence of specific, cis-actingsequences, origins of replication.

[0316] Considering that pericentric heterochromatin of mouse chromosomescontains thousands of short, simple repeats spanning 7-15 Mb, and thecentromere itself may also contain hundreds of kilobases, the existenceof a higher-order unit of replication seems probable. The observeduncontrolled intrachromosomal amplification restricted to a replicationinitiation region of the megachromosome is highly suggestive of arolling-circle type amplification, and provides additional evidence forthe presence of a replication origin in this region.

[0317] The finding that a specific replication initiation site occurs atthe boundaries of amplicons suggests that replication might play a rolein the amplification process. These results suggest that each ampliconof the megachromosome can be regarded as a huge megareplicon defined bya primary initiation site (megareplicator) containing “secondary”origins of replication. Fusion of replication bubbles from differentorigins of bi-directional replication (DePamphilis (1993) Ann. Rev.Biochem. 62:29-63) within the megareplicon could form a giantreplication bubble, which would correspond to the whole megareplicon. Inthe light of this, the formation of megabase-size amplicons can beaccommodated by a replication-directed amplification mechanism. In bothH and E-type amplifications, intrachromosomal multiplication of theamplicons was observed (see, above EXAMPLES), which is consistent withthe unequal sister chromatid exchange model. Induced or spontaneousunscheduled replication of a megareplicon in the constitutiveheterochromatin may also form new amplicon(s) leading to the expansionof the amplification or to the heterochromatic polymorphism of “normal”chromosomes. The “restoration” of the missing segment on the long arm ofthe megachromosome may well be the result of the re-replication of oneamplicon limited to one strand.

[0318] Taken together, without being bound by any theory, areplication-directed mechanism is a plausible explanation for theinitiation of large-scale amplifications in the centromeric regions ofmouse chromosomes, as well as for the de novo chromosome formations. Ifspecific (amplificator) sequences play a role in promoting theamplification process, sequences at the primary replication initiationsite (megareplicator) of the megareplicon are possible candidates.

[0319] Preliminary sequence data indicates the presence of highlyG+C-rich sequence elements less than 10 kb from the integratedheterologous “foreign” DNA in the megachromosome. These sequences mayrepresent the non-satellite DNA flanking of the A+T-rich satellite DNAblocks.

EXAMPLE 9

[0320] Generation of Chromosomes with Amplified Regions Derived fromMouse Chromosome 1

[0321] To show that the events described in EXAMPLES 2-7 are not uniqueto mouse chromosome 7 and to show that the EC7/3 cell line is notrequired for formation of these chromosomes, the experiments have beenrepeated using different initial cell lines and DNA fragments. Any cellor cell line should be amenable to use or can readily be determined tobe amenable or not.

[0322] A. Materials

[0323] The LP11 cell line was produced by the “scrape-loading”transfection method (Fechheimer et al. (1987) Proc. Natl. Acad. Sci.U.S.A. 84:8463-8467) using 25 μg plasmid DNA for 5×10⁶ recipient cells.LP11 cells were maintained in F-12 medium containing 3-15 μg/mlPuromycin (SIGMA).

[0324] B. Amplification in LP11 Cells

[0325] The large-scale amplification described in the above Examples isnot restricted to the transformed EC3/7 cell line or to the chromosome 7of mouse. In an independent transformation experiment, using aselectable puromycin construct pPuroTel, an LMTK⁻ cell line (LP11) wasestablished, carrying chromosome(s) with amplified chromosome segmentsof different lengths (˜150-600 Mb). Cytological analysis of the LP11cells indicated that the amplification occurred in the pericentricregion of the long arm of a submetacentric chromosome formed byRobertsonian translocation. This chromosome arm was identified byG-banding as chromosome 1. C-banding and in situ hybridization withmouse major satellite DNA probe showed that an E-type amplification hadoccurred: the newly formed region was composed of an array ofeuchromatic chromosome segments containing different amounts ofheterochromatin. The size and C-band pattern of the amplified segmentswere heterogeneous. In several cells, the number of these amplifiedunits exceeded 50; single-cell subclones of LP 11 cell lines, however,carry stable marker chromosomes with 10-15 segments and constant C-bandpatterns.

EXAMPLE 10

[0326] Purification of Artificial Chromosomes

[0327] I. Cell Sorting Based on Base Composition and Size

[0328] A. Cell Lines

[0329] 1B3 mouse-hamster-human hybrid cells (see, FIG. 4) carrying themegachromosome or the truncated megachromosome were grown in F-12 mediumsupplemented with 10% fetal calf serum, 150 μg/ml hygromycin B and 400μg/ml G418. GHB-42 (a cell line recloned from G3D5) mouse-hamster hybridcells carrying the megachromosome and the mini-chromosome were culturedin F-12 medium containing 10% fetal calf serum, 150 μg/ml hygromycin Band 400 μg/ml G418. The doubling time of both cell lines was about 24hours.

[0330] B. Chromosome Isolation

[0331] To accumulate mitotic cells, 5 μg/ml colchicine was added for 12hours to the cultures. Mitotic cells were then harvested by gentlepipetting of the medium on the layer cells. The mitotic index obtainedwas 60-80%. The mitotic cells by were collected by selective detachment.The cells were sedimented by centrifugation of 200 g for 10 minutes.

[0332] Two procedures were used to prepare metaphase chromosomes fromthese cells, one based on polyamine buffer system. (Cram et al. (1990)Methods in Cell Biology 33:377-382) and the other on modified hexyleneglycol buffer system (Hadlaczky et al. (1982) Chromosoma 86:643-65).

[0333] 1. Polyamine Procedure

[0334] In the polyamine procedure, about 10⁷ mitotic cells wereincubated in 10 ml hypotonic buffer (75 mM KCl, 0.2 mM spermine, 0.5 mMspermidine) for 10 minutes at room temperature to swell the cells. Thecells were then centrifuged at 100 g for 8 minutes. The cell pellet wasdrained carefully and about 10⁷ cells were resuspended in 1 ml polyaminebuffer (15 mM Tris-HCl, 20 mM NaCl, 80 mM KCl, 2 mM EDTA, 0.5 mM EGTA,14 mM β-mercaptoethanol, 0.1% digitonin, 0.2 mM Spermine, 0.5 mMspermidine). Chromosomes were then released by gently drawing the cellsuspension up and expelling it through a 22 G needle attached to a 3 mlplastic syringe. The chromosome concentration was about 1-3×10⁸chromosomes/ml.

[0335] 2. Hexylene Glycol Buffer System

[0336] In the second procedure, about 8×10⁶ mitotic cells wereresuspended in 10 ml glycine-hexylene glycol buffer (100 mM glycine, 1%hexylene glycol, pH 8.4-8.6 adjusted with saturated Ca-hydroxidesolution) and incubated for 10 minutes at 37° C., followed bycentrifugation for 10 minutes to pellet the nuclei. The supernatant wascentrifuged again at 200 g for 20 minutes to pellet the chromosomes.Chromosomes were resuspended in 1 ml isolation buffer/1-3×10⁸chromosomes.

[0337] C. Staining of Chromosomes with DNA Specific Dyes

[0338] Subsequent to isolation, the chromosome preparation was stainedwith Hoechst 33258 at 6 μg/ml and chromocycin A3 at 200 μg/ml. Fifteenminutes prior to analysis, 25 mM Na-sulphite and 10 mM Na-citrate wereadded to the chromosome suspension.

[0339] D. Flow Sorting of Chromosomes

[0340] Chromosomes in suspension were passed through a dual-laser cellsorter (FACStar Plus and FAXStar Vantage Becton DickinsonImmunocytometry System) in which two lasers were set to excite the dyesseparately, allowing a bivariate analysis of the chromosome by size andbase-pair composition. Because of the difference between the basecomposition of the MACs and the other chromosomes and the resultingdifference in interaction with the dyes, as well as size differences,the artificial chromosomes were separated from the other chromosomes.

[0341] E. Storage of the Sorted Artificial Chromosomes

[0342] The sorted chromosomes are stored in GH buffer (100 mM glycine,1% hexylene glycol pH 8.4-8.6 adjusted with saturated Ca-hydroxidesolution (see, e.g., Hadlaczky et al. (1982) Chromosoma 86:643-659) forone day and embedded by centrifugation into agarose. The sortedchromosomes were centrifuged into an agarose bed and the plugs arestored in 500 mM EDTA at 4° C. They are stored for microinjection in 30%glycerol at −20° C.

[0343] F. Quality Control

[0344] 1. Analysis of the Purity

[0345] The purity of the sorted chromosomes was checked by fluorescencein situ hybridization (FISH) with biotin labeled mouse satellite DNAprobe (see, Hadlaczky et al. (1991) Proc. Natl. Acad. Sci. U.S.A.88:8106-8110. Purity of the sorted chromosomes was 97-99%.

[0346] 2. Characteristics of the Sorted Chromosomes

[0347] Pulsed field gel electrophoresis and Southern hybridization werecarried out to determine the size distribution of the DNA content of thesorted artificial chromosomes.

[0348] C. Functioning of the Artificial Chromosomes

[0349] To check whether their activity is preserved, the artificialchromosomes are microinjected into primary cells, somatic cells and stemcells.

[0350] II. Sorting of Mammalian Artificial Chromosome ContainingMicrocells

[0351] A. Micronucleation

[0352] Cells were grown to 80-90% confluency in 4 T150 flasks. Colcemidwas added to a final concentration of 0.06 μg/ml, and then incubatedwith the cells at 37° C. for 24 hours.

[0353] B. Enucleation

[0354] Ten μg/ml cytochalasin B was added and the resulting microcellswere centrifuged the at 15,000 rpm for 70 minutes at 28-33° C.

[0355] C. Purification of Microcells by Filtration

[0356] The microcells were purified using Swinnex filter units andNucleopore filters (5 μm and 3 μm).

[0357] D. Staining and Sorting Microcells

[0358] As above, the cells were stained Hoechst and chromomycin A3 dyes.The microcells were sorted by cell sorter to isolate the microcells thatcontain the mammalian artificial chromosome.

[0359] E. Fusion

[0360] The microcells that contain the artificial chromosome are fusedto selected primary cells, somatic cells, embryonic stem cells togenerate transgenic animals for gene therapy purposes, and other cellsto deliver the chromosomes to the cells.

EXAMPLE 11

[0361] Introduction of Mammalian Artificial Chromosomes into InsectCells

[0362] Insect cells should be useful hosts for MACs, particularly forproduction of gene products for a number of reasons, including:

[0363] 1. A mammalian artificial chromosome provides extra genomicspecific integration site for introduction of genes encoding proteins ofinterest (reduced chance of mutation in production system).

[0364] 2. The large size of artificial chromosome permits megabase sizeDNA integration so that genes encoding an entire pathway leading to aprotein or nonprotein of therapeutic value, such as an alkaloid(digitalis, morphine, taxol).

[0365] 3. Amplification of genes encoding useful proteins can beaccomplished in the artificial mammalian chromosome to obtain higherprotein yields in insect cells.

[0366] 4. Insect cells support required post translational modifications(glycosylation, phosphorylation) essential for protein biologicalfunction.

[0367] 5. Insect cells do not support mammalian viruses—eliminatescross-contamination of product with human infectious agents.

[0368] 6. The ability to introduce chromosomes, circumvents traditionalrecombinant baculovirus systems for production of nutritional,industrial or medicinal proteins in insect cell systems.

[0369] 7 The low temperature optimum for insect cell growth (28° C.)permits reduced energy cost of production.

[0370] 8. Serum free growth medium for insect cells will result in lowerproduction costs.

[0371] 9. Artificial chromosome containing cells can be storedindefinitely at low temperature.

[0372] 10. Insect larvae will serve as biological factories for theproduction of nutritional, medicinal or industrial proteins bymicroinjection of fertilized insect eggs.

[0373] A. Demonstration that Insect Cells Recognize Mammalian Promoters

[0374] Gene constructs containing a mammalian promoter, such as CMVlinked to DNA encoding a detectable marker gene fusions (Renillaluciferase gene (see, e.g., U.S. Pat. No. 5,292,658 for a description ofDNA encoding the Renilla luciferase, and plasmid pTZrLuc-1, which canprovide the starting material for construction of such vectors, see SEQID NO. 10) and also including the simian virus 40 (SV40) promoteroperably linked to the beta galactosidase gene) was introduced into thecells of two species Trichoplusia ni (cabbage looper) and Bombyx mori(silk worm).

[0375] After transferring the constructs into the insect cell lineseither by electroporation or by microinjection, expression of the markergenes was detected after a 24 h incubation. In each case a positiveresult was obtained in the samples containing the genes which was absentin samples in which the genes were omitted. In addition, a β-actinpromoter-Renilla luciferase fusion was introduced into the T. ni and B.mori cells yielding light emission. Thus, mammalian promoters functionto direct expression of these marker genes in insects. Therefore, MACsare candidates for expression of heterologous genes in insect cells.

[0376] B. Construction of Vectors for Use in Insect Cells and Fusionwith Mammalian Cells

[0377] 1. Transform LMTK⁻ cells with expression vector with:

[0378] a. B. mori β-actin promoter—Hyg^(r) selectable marker gene forinsect cells, and.

[0379] b. SV40 or CMV promoters controlling a puromycin^(r) selectablemarker gene for mammalian cells.

[0380] 2. Detect expression of the mammalian promoter in LMTk cells(puromycin^(r) LMTk cells)

[0381] 3. Use pur^(r) cells in fusion experiments with Bombyx andTrichoplusia cells, select Hyg^(r) cells.

[0382] C. Insertion of the MACs into Insect Cells

[0383] These experiments are designed to detect expression of adetectable marker gene (such as β-gal expressed under the control of amammalian promoter, such as pSV40) located on a MAC. Data indicate thatβ-gal was expressed.

[0384] Insect cells of each species are fused with mammalian cellscontaining either the mini chromosome (EC3/7C5) or the mini and themegachromosome (such as GHB-42, which is a cell line recloned from G3D5)or a cell line that carries only the megachromosome (such as H1D3 or aredone therefrom). Fusion is carried out as follows:

[0385] 1. mammalian+insect cells (50/50%) in log phase growth are mixed;

[0386] 2. calcium/PEG cell fusion: (10 min-0.5 h);

[0387] 3. heterokaryons (+72 h) are selected.

[0388] The following selection conditions to select for insect cellsthat contain a MAC can be used: (+=positive selection; −=negativeselection):

[0389] 1. growth at 28° C. (+ insect cells, − mammalian cells);

[0390] 2. Graces insect cell medium (SIGMA) (− mammalian cells);

[0391] 3. no exogenous CO₂ (− mammalian cells); and/or

[0392] 4. antibiotic selection (Hyg or G418) (+ transformed insectcells).

[0393] Immediately following the fusion protocol, many heterokaryons(fusion events) are observed between the mammalian and each species ofinsect cells (up to 90% heterokaryons). After growth (2+weeks) on insectmedium containing G418 and/or hygromycin at selection levels used forselection of transformed mammalian cells, individual colonies aredetected growing on the fusion plates. By virtue of selection for theantibiotic resistance conferred by the MAC and selection for insectcells, these colonies should contain MACs.

EXAMPLE 12

[0394] Preparation of the Chromosome Fragmentation Vector and OtherVectors for Targeted Integration of DNA into MACs

[0395] Fragmentation of the megachromosome, should ultimately result insmaller stable chromosomes that contain about 15 Mb to 50 Mb that willbe easily manipulated for use as vectors. Vectors to effect suchfragmentation should also aid in determination and identification of theelements required for preparation of an in vitro-produced artificialchromosome.

[0396] Reduction in the size of the megachromosome can be achieved in anumber of different ways including: stress treatment, such as bystarvation, or cold or heat treatment; treatment with agents thatdestabilize the genome or nick DNA, such as BrdU, coumarin, EMS andothers; treatment with ionizing radiation (see, e.g., Brown (1992) Curr.Opin. Genes Dev. 2:479-486); and telomere-directed in vivo chromosomefragmentation (see, e.g., Far et al. (1995) EMBO J. 14:5444-5454).

[0397] A. Preparation Vectors for Fragmentation of the ArtificialChromosome and Also for Targeted Integration of Selected Gene Products

[0398] 1. Construction of pTEMPUD

[0399] Plasmid pTEMPUD (see, FIG. 5) is a mouse homologous recombination“killer” vector for in vivo chromosome fragmentation, and also forinducing large-scale amplification via site specific integration. Withreference to FIG. 5, the PstI to SalI fragment was derived frompBabe-puro retroviral vector (see, Morgenstern et al. (1990) NucleicAcids Res. 18:3587-3596). This fragment contains DNA encoding ampicillinresistance, the pUC origin of replication, and the puromycin N-acetyltransferase gene under control of the SV40 early promoter. The URA3portion comes from the pYAC5 cloning vector (SIGMA). URA3 was cut out ofpYAC5 with SalI-XhoI digestion, cloned into pNEB193 (New EnglandBiolabs), which was then cut with EcoRI-SalI and ligated to the SalIsite of pBabepuro to produce pPU.

[0400] A 1293 bp fragment (see SEQ ID NO. 1) encoding the mouse majorsatellite, was isolated as an EcoRI fragment from a DNA library producedfrom mouse LMTK⁻ fibroblast cells and inserted into the EcoRI site ofpPU to produce pMPU.

[0401] The TK promoter driven diphtheria toxin gene (DT-A) was derivedfrom pMC1DT-A (see, Maxwell et al. (1986) Cancer Res. 46:4660-4666) byBglII-XhoI digestion and cloned into the pMC1neo poly A expressionvector (STRATAGENE, La Jolla, Calif.) by replacing the neo codingsequence. The TK promoter, DT-A gene and poly A sequence were removedfrom this vector, cohesive ends were filled with Klenow and theresulting fragment blunt end-ligated and ligated into the SnaB1 (TACGTA)of pMPU to produce pMPUD.

[0402] The Hutel 2.5 fragment (see SEQ ID NO. 3) was inserted at thePstI site (see the 6100 PstI-3625 PstI fragment on pTEMPUD) of pMPUD toproduce pTEMPUD. This fragment includes a human telomere. It includes aunique BglII site (see nucleotides 1042-1047 of SEQ ID NO. 3), whichwill be used as a site for introduction of a synthetic telomere thatwill include multiple repeats (80) of GGGATT with BamHI and BglII endsfor insertion into the BglII site which will then remain unique, sincethe BamHI overhang is compatible with the BglII site. Ligation of BamHIfragment to a BglII destroys the BglII site, so that only a single BglIIsite will remain. Selection for the unique BglII site insures that thesynthetic telomere will be inserted in the correct orientation. Theunique BglII site is the site at which the vector is linearized.

[0403] 2. Use of pTEMPUD for In Vivo Chromosome Fragmentation

[0404] Linearization of pTEMPUD by BglII results in a linear moleculewith a human telomere at one end. Integration of this linear fragmentinto the chromosome, such as the megachromosome in hybrid cells or anymouse chromosome, which is contains repeats of the mouse major satellitesequence, results integration of the selectable marker puromycin andcleavage of the plasmid by virtue of the telomeric end. The DT geneprevents that entire linear fragment from integrating by random events,since upon integration and expression it is toxic. Thus randomintegration will be toxic. Thus, site directed integration into thetargeted DNA will be selected. Such integration will produce fragmentedchromosomes.

[0405] The fragmented truncated chromosome with the new telomere willsurvive, and the other fragment without the centromere will be lost.Repeated in vivo fragmentations will ultimately result in selection ofthe smallest functioning minichromosome possible.

[0406] Thus this vector can be used to produce minichromosomes frommouse chromosomes, or to fragment the megachromosome.

[0407] 3. pTEMPhu and pTEMPhu3

[0408] Vectors that specifically target human chromosomes can beconstructed from pTEMPUD. These vectors can be used to fragment specifichuman chromosomes, depending upon the selected satellite sequence, toproduce human minichromosomes, and also to isolate human centromeres.

[0409] a. pTEMPhu

[0410] To render pTEMPUD suitable for fragmenting human chromosomes, themouse major satellite sequence is replaced with human satellitesequences. Unlike mouse chromosomes, each human chromosome has a uniquesatellite sequence. For example, the mouse major satellite has beenreplace with a human hexameric α-satellite (or alphoid satellite) DNAsequence. This sequence is an 813 bp fragment (nucleotide 232-1044 ofSEQ ID NO. 2) from clone pS12, deposited in the EMBL database underAccession number X60716, isolated from a human colon carcinoma cell lineColo320 (deposited under Accession No. ATCC CCL 220.1). The 813 bpalphoid fragment can be obtained from the pS12 clone by nucleic acidamplification using synthetic primers, which each contain an EcoRI site,as follows:

[0411] GGGGAATTCAT TGGGATGTTT CAGTTGA forward primer (SEQ ID NO. 4)

[0412] CGAAAGTCCCC CCTAGGAGAT CTTAAGGA reverse primer (SEQ ID NO. 5).

[0413] Digestion of the amplified product with EcoRI results in afragment with EcoRI ends that includes the human α-satellite sequence.This sequence is inserted into pTEMPUD in place of the EcoRI fragmentthat contains the mouse major satellite.

[0414] b. pTEMPhu3

[0415] In pTEMPhu3, the mouse major satellite sequence is replaced bythe 3 kb human chromosome 3-specific α-satellite from D3Z1 (depositedunder ATCC Accession No. 85434; see, also Yrokov (1989) Cytogenet. CellGenet. 51:1114).

[0416] 4. Use of the pTEMPHU3 to Induce Amplification on HumanChromosome #3

[0417] Each human chromosome contains unique chromosome-specific alphoidsequence. Thus, use of pTEMPHU3, which is targeted to the chromosome3-specific α-satellite can be introduced into human cells underselective conditions, whereby large scale amplification of thechromosome 3 centromeric region and production of a de novo chromosome.Such induced large-scale amplification provides a means for inducing denovo chromosome formation and also for in vivo cloning of defined humanchromosome fragments up to megabase size.

[0418] For example, the break-point in human chromosome #3 is on theshort arm near the centromere. this region is involved in renal cellcarcinoma formation. By targeting pTEMPhu3 to this region, the inducedlarge-scale amplification may contain this region, which can then becloned using the bacterial and yeast markers in the pTEMPhu3 vector.

[0419] The pTEMPhu3 cloning vector allows not only selection forhomologous recombinants, but also direct cloning of the integration sitein YACS. This vector can also be used to target human chromosome #3,preferably with a deleted short arm, in a mouse-human monochromosomalmicrocell hybrid line. Homologous recombinants can be screened bynucleic acid amplification (PCR) and amplification can be screened byDNA hybridization, Southern hybridization, and in situ hybridization.The amplified region can be cloned into YAC. This vector and thesemethods also permit a functional analysis of cloned chromosome regionsby reintroducing the cloned amplified region into mammalian cells.

[0420] B. Preparation of Libraries in YAC Vectors for Cloning ofCentromeres and Identification of Functional Chromosomal Units

[0421] Another method that may be used to obtain smaller-sizedfunctional mammalian artificial chromosome units and to clonecentromeric DNA involves screening of mammalian DNA YAC vector-basedlibraries and functional analysis of potential positive clones in atransgenic mouse model system. A mammalian DNA library is prepared in aYAC vector, such as YRT2 (see Schedl et al. (1993) Nuc. Acids Res.21:4783-4787), which contains the murine tyrosinase gene. The library isscreened for hybridization to mammalian telomere and centromere sequenceprobes. Positive clones are isolated and microinjected into pronuclei offertilized oocytes of NMRI/Han mice following standard techniques. Theembryos are then transferred into NMRI/Han foster mothers. Expression ofthe tyrosinase gene in transgenic offspring confers an identifiablephenotype (pigmentation). The clones that give rise totyrosinase-expressing transgenic mice are thus confirmed as containingfunctional mammalian artificial chromosome units.

[0422] Alternatively, fragments of SATACs may be introduced into the YACvectors and then introduced into pronuclei of fertilized oocytes ofNMRI/Han mice following standard techniques as above. The clones thatgive rise to tyrosinase-expressing transgenic mice are thus confirmed ascontaining functional mammalian artificial chromosome units,particularly centromeres.

[0423] C. Incorporation of Heterologous Genes into Mammalian ArtificialChromosomes Through the Use of Homology Targeting Vectors

[0424] As described above, the use of mammalian artificial chromosomesfor expression of heterologous genes obviates certain negative effectsthat may result from random integration of heterologous plasmid DNA intothe recipient cell genome. An essential feature of the mammalianartificial chromosome that makes it a useful tool in avoiding thenegative effects of random integration is its presence as anextra-genomic gene source in recipient cells. Accordingly, methods ofspecific, targeted incorporation of heterologous genes exclusively intothe mammalian artificial chromosome, without extraneous randomintegration into the genome of recipient cells, are desired forheterologous gene expression from a mammalian artificial chromosome.

[0425] One means of achieving site-specific integration of heterologousgenes into artificial chromosomes is through the use of homologytargeting vectors. The heterologous gene of interest in subcloned into atargeting vector which contains nucleic acid sequences that arehomologous to nucleotides present in the artificial chromosome. Thevector is then introduced into cells containing the artificialchromosome for specific site-directed integration into the artificialchromosome through a recombination event at sites of homology betweenthe vector and the chromosome. The homology targeting vectors may alsocontain selectable markers for ease of identifying cells that haveincorporated the vector into the artificial chromosome as well as lethalselection genes that are expressed only upon extraneous integration ofvector into the recipient cell genome. Two exemplary homology targetingvectors, λCF-7 and pλCF-7-DTA, are described below.

[0426] 1. Construction of Vector λCF-7

[0427] Vector λCF-7 contains the cystic fibrosis transmembraneconductance regulator (CFTR) gene as an exemplary therapeuticmolecule-encoding nucleic acid that may be incorporated into mammalianartificial chromosomes for use in gene therapy applications. Thisvector, which also contains the puromycin-resistance gene as aselectable marker, as well as the Saccharomyces cerevisiae ura3 gene(orotidine-5-phosphate decarboxylase), was constructed in a series ofsteps as follows.

[0428] a. Construction of pURA

[0429] Plasmid pURA was prepared by ligating a 2.6-kb SalI/XhoI fragmentfrom the yeast artificial chromosome vector pYAC5 (Sigma; see also Burkeet al. (1987) Science 236:806-812 for a description of YAC vectors aswell as GenBank Accession No. U01086 for the complete sequence of pYAC5)containing the S. cerevisiae ura3 gene with a 3.3-kb SalI/SmaI fragmentof pHyg (see, e.g., U.S. Pat. Nos. 4,997,764, 4,686,186 and 5,162,215,and the description above). Prior to ligation the XhoI end was treatedwith Klenow polymerase for blunt end ligation to the SmaI end of the 3.3kb fragment of pHyyg. Thus, pURA contains the S. cerevisiae ura3 gene,and the E. coli ColE1 origin of replication and theampicillin-resistance gene. The uraE gene is included to provide a meansto recover the integrated construct from a mammalian cell as a YACclone.

[0430] b. Construction of pUP2

[0431] Plasmid pURA was digested with SalI and ligated to a 1.5-kb SalIfragment of pCEPUR. Plasmid pCEPUR is produced by ligating the 1.1 kbSnaBI-NhaI fragment of pBabe-puro (Morgenstern et al. (1990) Nucl. AcidsRes. 18:3587-3596; provided by Dr. L. Székely (Microbiology andTumorbiology Center, Karolinska Institutet, Stockholm); see, alsoTonghua et al. (1995) Chin. Med. J. (Beijing, Engl. Ed.) 108:653-659;Couto et al. (1994) Infect. Immun. 62:2375-2378; Dunckley et al. (1992)FEBS Lett. 296:128-34; French et al. (1995) Anal. Biochem. 228:354-355;Liu et al. (1995) Blood 85:1095-1103; International PCT application Nos.WO 9520044; WO 9500178, and WO 9419456) to the NheI-NruI fragment ofpCEP4 (Invitrogen).

[0432] The resulting plasmid, pUP2, contains the all the elements ofpURA plus the puromycin-resistance gene linked to the SV40 promoter andpolyadenylation signal from pCEPUR.

[0433] c. Construction of pUP-CFTR

[0434] The intermediate plasmid pUP-CFTR was generated in order tocombine the elements of pUP2 into a plasmid along with the CFTR gene.First, a 4.5-kb SalI fragment of pCMV-CFTR that contains theCFTR-encoding DNA (see, also, Riordan et al. (1989) Science245:1066-1073, U.S. Pat. No. 5,240,846, and Genbank Accession No. M28668for the sequence of the CFTR gene) containing the CFTR gene only wasligated to XhoI-digested pCEP4 (Invitrogen and also described herein) inorder to insert the CFTR gene in the multiple cloning site of theEpstein Barr virus-based (EBV) vector pCEP4 (Invitrogen, San Diego,Calif.; see also Yates et al. (1985) Nature 313:812-815; see, also U.S.Pat. No. 5,468,615) between the CMV promoter and SV40 polyadenylationsignal. The resulting plasmid was designated pCEP-CFTR. PlasmidpCEP-CFTR was then digested with SalI and the 5.8-kb fragment containingthe CFTR gene flanked by the CMV promoter and SV40 polyadenylationsignal was ligated to SalI-digested pUP2 to generate pUP-CFTR. Thus,pUP-CFTR contains all elements of pUP2 plus the CFTR gene linked to theCMV promoter and SV40 polyadenylation signal.

[0435] d. Construction of λCF-7

[0436] Plasmid pUP-CFTR was then linearized by partial digestion withEcoRI and the 13 kb fragment containing the CFTR gene was ligated withEcoRI-digested Charon 4A λ (see Blattner et al. (1977) Science 196:161;Williams and Blattner (1979) J. Virol. 29:555 and Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual, Second Ed., Cold Spring HarborLaboratory Press, Volume 1, Section 2.18, for descriptions of Charon4Aλ). The resulting vector, λCF8, contains the Charon 4Aλ bacteriophageleft arm, the CFTR gene linked to the CMV promoter and SV40polyadenylation signal, the ura3 gene, the puromycin-resistance genelinked to the SV40 promoter and polyadenylation signal, the thymidinekinase promoter (TK), the ColE1 origin of replication, the ampicillinresistance gene and the Charon 4Aλ bacteriophage right arm. The λCF8construct was then digested with XhoI and the resulting 27.1 kb wasligated to the 0.4 kb XhoI/EcoRI fragment of pJBP86 (described below),containing the SV40 polyA signal and the EcoRI-digested Charon 4A λright arm. The resulting vector λCF-7 contains the Charon 4A λ left arm,the CFTR encoding DNA linked to the CMV promoter and SV40 polyA signal,the ura3 gene, the puromycin resistance gene linked to the SV40 promoterand polyA signal and the Charon 4A λ right arm. The λ DNA fragmentsprovide sequences homologous to nucleotides present in the exemplaryartificial chromosomes.

[0437] The vector is then introduced into cells containing theartificial chromosomes exemplified herein. Accordingly, when the linearλCF-7 vector is introduced into megachromosome-carrying fusion celllines, such as described herein, it will be specifically integrated intothe megachromosome through recombination between the homologousbacteriophage λ sequences of the vector and the artificial chromosome.

[0438] 2. Construction of Vector λCF-7-DTA

[0439] Vector λCF-7-DTA also contains all the elements contained inλCF-7, but additionally contains a lethal selection marker, thediphtheria toxin-A (DT-A) gene as well as the ampicillin-resistance geneand an origin of replication. This vector was constructed in a series ofsteps as follows.

[0440] a. Construction of pJBP86

[0441] Plasmid pJBP86 was used in the construction of λCF-7, above. A1.5-kb SalI fragment of pCEPUR containing the puromycin-resistance genelinked to the SV40 promoter and polyadenylation signal was ligated toHindIII-digested pJB8 (see, e.g., Ish-Horowitz et al. (1981) NucleicAcids Res. 9:2989-2998; available from ATCC as Accession No. 37074;commercially available from Amersham, Arlington Heights, Ill.). Prior toligation the SalI ends of the 1.5 kb fragment of pCEPUR and the HindIIIlinearized pJB8 ends were treated with Klenow polymerase beforeligation. The resulting vector pJBP86 contains the puromycin resistancegene linked to the SV40 promoter and polyA signal, the 1.8 kb COS regionof Charon 4Aλ, the ColE1 origin of replication and the ampicillinresistance gene.

[0442] b. Construction of pMEP-DTA

[0443] A 1.1-kb XhoI/SalI fragment of pMC1-DT-A (see, e.g., Maxwell etal. (1986) Cancer Res. 46:4660-4666) containing the diphtheria toxin-Agene was ligated to XhoI-digested pMEP4 (Invitrogen, San Diego, Calif.)to generate pMEP-DTA. To produce pMC1-DT-A, the coding region of the DTAgene was isolated as a 800 bp PstI/HindIII fragment from p2249-1 andinserted into pMC1neopolyA (pMC1 available from Stratagene) in place ofthe neo gene and under the control of the TK promoter. The resultingconstruct pMC1DT-A was digested with HindIII, the ends filled by Klenowand SalI linkers were ligated to produce a 1061 bp TK-DTA gene cassettewith an XhoI end (5′) and a SalI end containing the 270 bp TK promoterand the ˜790 bp DT-A fragment. This fragment was ligated intoXhoI-digested pMEP4.

[0444] Plasmid pMEP-DTA thus contains the DT-A gene linked to the TKpromoter and SV40, ColE1 origin of replication and theampicillin-resistance gene.

[0445] c. Construction of pJB83-DTA9

[0446] Plasmid pJB8 was digested with HindIII and ClaI and ligated withan oligonucleotide (see SEQ ID NOs. 7 and 8 for the sense and antisensestrands of the oligonucleotide, respectively) to generate pJB83. Theoligonucleotide that was ligated to ClaI/HindIII-digested pJB8 containedthe recognition sites of SwaI, PacI and SrfI restriction endonucleases.These sites will permit ready linearization of the pλCF-7-DTA construct.

[0447] Next, a 1.4-kb XhoI/SalI fragment of pMEP-DTA, containing theDT-A gene was ligated to SalI-digested pJB83 to generate pJB83-DTA9.

[0448] d. Construction of λCF-7-DTA

[0449] The 12-bp overhangs of λCF-7 were removed by Mung bean nucleaseand subsequent T4 polymerase treatments. The resulting 41.1-kb linearλCF-7 vector was then ligated to pFB83-DTA9 which had been digested withClaI and treated with T4 polymerase. The resulting vector, λCF-7-DTA,contains all the elements of λCF-7 as well as the DT-A gene linked tothe TK promoter and the SV40 polyadenylation signal, the 1.8 kB Charon4A λ COS region, the ampicillin-resistance gene (from pJB83-DTA9) andthe Col E1 origin of replication (from pJB83-DT9A).

[0450] 3. pMCT-RUC,

[0451] Plasmid pMCT-RUC (14 kbp) was constructed for site-specifictargeting of the Renilla luciferase (see, e.g., U.S. Pat. Nos. 5,292,658and 5,418,155 for a description of DNA encoding Renilla luciferase, andplasmid pTZrLuc-1, which can provide the starting material forconstruction of such vectors) gene to a mammalian chromosome. Therelevant features of this plasmid are the Renilla luciferase gene undertranscriptional control of the human cytomegalovirus immediate-earlygene enhancer/promoter; the hygromycin gene under the transcriptionalcontrol of the thymidine kinase promoter; and a unique HpaI site is forlinearizing the plasmid.

[0452] This construct was introduced into C5 cells (see, Lorenz et al.(1996) J. Biolum. Chemilum. 11:31-37). C5 mouse fibroblasts weremaintained as a monolayer (see, Gluzman (1981) Cell 23:175-183). Cellsat 50% confluency in 100 mm Petri dishes were used for calcium phosphatetransfection (see, Harper et al. (1981) Chromosoma 83:431-439) using 10μg of linearized pMCT-RUC per plate. Colonies originating from singletransfected cells were isolated and maintained in F-12 medium containinghygromycin (300 μg/mL) and 10% fetal bovine serum. Cells were grown in100 mm Petri dishes prior to the Renilla luciferase assay.

[0453] The Renilla luciferase assay was performed (see, e.g., Matthewset al. (1977) Biochemistry 16:85-91). Hygromycin-resistant cell linesobtained after transfection of mouse fibroblasts with linearized plasmidpMCT-RUC (“B” cell lines) were grown to 100% confluency for measurementsof light emission in vivo and in vitro. Light emission was measured invivo after about 30 generations as follows: growth medium was removedand replaced by 1 mL RPMI 1640 containing coelenterazine (1 mmol/L finalconcentration). Light emission from cells was then visualized by placingthe Petri dishes in a low light video image analyzer (HamamatsuArgus-100). An image was formed after 5 min. of photon accumulationusing 100% sensitivity of the photon counting tube. For measuring lightemission in vitro, cells were trypsinized and harvested from one Petridish, pelleted, resuspended in 1 mL assay buffer (0.5 mol/L NACl, 1mmol/L EDTA, 0.1 mol/L potassium phosphate, pH 7.4) and sonicated on icefor 10 s. Lysates were than assayed in a Turner TD-20e lukminometer for10 s after rapid injection of 0.5 mL of 1 mmol/L coelenterazine, and theaverage value of light emission was recorded as LU (1 LU=1.6×106 hu/sfor this instrument).

[0454] Independent cell lines of mouse fibroblasts transfected withlinearized plasmid pMCT-RUC showed different levels of Renillaluciferase activity. Similar differences in light emission were observedwhen measurements were performed on lysates of the same cell lines. Thisvariation in light emission was probably due to a position effectresulting from the random integration of plasmid pMCT-RUC into the mousegenome, since enrichment for site targeting of the luciferase gene wasnot performed in this experiment.

[0455] D. Protein Secretion Targeting Vectors

[0456] Isolation of heterologous proteins produced intracellularly inmammalian cell expression systems requires cell disruption underpotentially harsh conditions and purification of the recombinant proteinfrom cellular contaminants. The process of protein isolation may begreatly facilitated by secretion of the recombinantly produced proteininto the extracellular medium where there are fewer contaminants toremove during purification. Therefore, secretion targeting vectors havebeen constructed for use with the mammalian artificial chromosomesystem.

[0457] A useful model vector for demonstrating production and secretionof heterologous protein in mammalian cells contains DNA encoding areadily detectable reporter protein fused to an efficient secretionsignal that directs transport of the protein to the cell membrane andsecretion of the protein from the cell. Vectors pLNCX-ILRUC andpLNCX-ILRUCλ, described below, are examples of such vectors. Thesevectors contain DNA encoding an interleukin-2 (IL2) signalpeptide-Renilla reniformis luciferase fusion protein. The IL-2 signalpeptide (encoded by the sequence set forth in SEQ ID NO. 9) directssecretion of the luciferase protein, to which it is linked, frommammalian cells. Upon secretion from the host mammalian cell, the IL-2signal peptide is cleaved from the fusion protein to deliver mature,active, luciferase protein to the extracellular medium. Successfulproduction and secretion of this heterologous protein can be readilydetected by performing luciferase assays which measure the light emittedupon exposure of the medium to the bioluminescent luciferin substrate ofthe luciferase enzyme.

[0458] 1. Construction of Protein Secretion Vector pLNCX-ILRUC

[0459] Vector pLNCX-ILRUC contains a human IL-2 signal peptide-R.reniformis fusion gene linked to the human cytomegalovirus (CMV)immediate early promoter for constitutive expression of the gene inmammalian cells. The construct was prepared as follows.

[0460] a. Preparation of the IL-2 Signal Sequence-Encoding DNA

[0461] A 69-bp DNA fragment containing DNA encoding the human IL-2signal peptide was obtained through nucleic acid amplification, usingappropriate primers for IL-2, of an HEK 293 cell line (see, e.g., U.S.Pat. No. 4,518,584 for an IL-2 encoding DNA; see, also SEQ ID NO. 9; theIL-2 gene and corresponding amino acid sequence is also provided in theGenbank Sequence Database as accession nos. K02056 and J00264). Thesignal peptide includes the first 20 amino acids shown in thetranslations provided in both of these Genbank entries and in SEQ ID NO.9. The corresponding nucleotide sequence encoding the first 20 aminoacids is also provided in these entries (see, e.g., nucleotides 293-52of accession no. K02056 and nucleotides 478-537 of accession no.J00264), as well as in SEQ ID NO. 9. The amplification primers includedan EcoRI site (GAATTC) that for subcloning of the DNA fragment intoEcoRI-digested pGEMT (Promega). The forward primer is set forth in SEQID NO. 11 and the sequence of the reverse primer is set forth in SEQ IDNO. 12.

[0462] TTTGAATTCATGTACAGGATGCAACTCCTG forward (SEQ ID NO. 11)

[0463] TTTGAATTCAGTAGGTGCACTGTTTGTGAC reverse (SEQ ID NO. 12)

[0464] b. Preparation of the R. reniformis Luciferase-Encoding DNA

[0465] The initial source of the R. reniformis luciferase gene wasplasmid pLXSN-RUC. Vector pLXSN (see, e.g., U.S. Pat. Nos. 5,324,655,5,470,730, 5,468,634, 5,358,866 and Miller et al. (1989) Biotechniques7:980) is a retroviral vector capable of expressing heterologous DNAunder the transcriptional control of the retroviral LTR; it alsocontains the neomycin-resistance gene operatively linked for expressionto the SV40 early region promoter. The R. reniformis luciferase gene wasobtained from plasmid pTZrLuc-1 (see, e.g., U.S. Pat. No. 5,292,658; seealso the Genbank Sequence Database Accession No. M63501; and see alsoLorenz et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88:4438-4442) and isshown as SEQ ID NO. 10. The 0.97 kb EcoRI/SmaI fragment of pTZrLuc-1contains the coding region of the Renilla luciferase-encoding DNA.Vector pLXSN was digested with and ligated with the luciferase genecontained on a pLXSN-RUC, which contains the luciferase gene locatedoperably linked to the viral LTR and upstream of the SV40 promoter,which directs expression of the neomycin-resistance gene.

[0466] c. Fusion of DNA Encoding the IL-2 Signal Peptide and the R.reniformis Luciferase Gene to Yield pLXSN-ILRUC

[0467] The pGEMT vector containing the IL-2 signal peptide-encoding DNAdescribed in 1.a. above was digested with EcoRI, and the resultingfragment encoding the signal peptide was ligated to EcoRI-digestedpLXSN-RUC. The resulting plasmid, called pLXSN-ILRUC, contains the IL-2signal peptide-encoding DNA located immediately upstream of the R.reniformis gene in pLXSN-RUC. Plasmid pLXSN-ILRUC was then used as atemplate for nucleic acid amplification of the fusion gene in order toadd a SmaI site at the 3′ end of the fusion gene. The amplificationproduct was subcloned into EcoRI/SmaI-digested pGEMT (Promega) togenerate ILRUC-pGEMT.

[0468] d. Introduction of the Fusion Gene into a Vector ContainingControl Elements for Expression in Mammalian Cells

[0469] Plasmid ILRUC-pGEMT was digested with KspI and SmaI to release afragment containing the IL-2 signal peptide-luciferase fusion gene whichwas ligated to HpaI-digested pLNCX. Vector pLNCX (see, e.g., U.S. Pat.Nos. 5,324,655 and 5,457,182; see, also Miller and Rosman (1989)Biotechniques 7:980-990) is a retroviral vector for expressingheterologous DNA under the control of the CMV promoter; it also containsthe neomycin-resistance gene under the transcriptional control of aviral promoter. The vector resulting from the ligation reaction wasdesignated pLNCX-ILRUC. Vector pLNCX-ILRUC contains the IL-2 signalpeptide-luciferase fusion gene located immediately downstream of the CMVpromoter and upstream of the viral 3′ LTR and polyadenylation signal inpLNCX. This arrangement provides for expression of the fusion gene underthe control of the CMV promoter. Placement of the heterologousprotein-encoding DNA (i.e., the luciferase gene) in operative linkagewith the IL-2 signal peptide-encoding DNA provides for expression of thefusion in mammalian cells transfected with the vector such that theheterologous protein is secreted from the host cell into theextracellular medium.

[0470] 2. Construction of Protein Secretion Targeting VectorpLNCX-ILRUCλ

[0471] Vector pLNCX-ILRUC may be modified so that it can be used tointroduce IL-2 signal peptide-luciferase fusion gene into a mammalianartificial chromosome in a host cell. To facilitate specificincorporation of the pLNCX-ILRUC expression vector into a mammalianartificial chromosome, nucleic acid sequences that are homologous tonucleotides present in the artificial chromosome are added to the vectorto permit site directed recombination.

[0472] Exemplary artificial chromosomes described herein contain lambdaphage DNA. Therefore, protein secretion targeting vector pLNCX-ILRUCλwas prepared by addition of lambda phage DNA (from Charon 4A arms) toproduce the secretion vector pLNCX-ILRUC.

[0473] 3. Expression and Secretion of R. reniformis Luciferase fromMammalian Cells

[0474] a. Expression of R. reniformis Luciferase Using pLNCX-ILRUC

[0475] Mammalian cells (LMTK⁻ from the ATCC) were transientlytransfected with vector pLNCX-ILRUC (˜10 μg) by electroporation (BIORAD,performed according to the manufacturer's instructions). Stabletransfectants produced by growth in G418 for neo selection have alsobeen prepared.

[0476] Transfectants were grown and then analyzed for expression ofluciferase. To determine whether active luciferase was secreted from thetransfected cells, culture media were assayed for luciferase by additionof coelentrazine (see, e.g., Matthews et al. (1977) Biochemistry16:85-91).

[0477] The results of these assays establish that vector pLNCX-ILRUC iscapable of providing constitutive expression of heterologous DNA inmammalian host cells. Furthermore, the results demonstrate that thehuman IL-2 signal peptide is capable of directing secretion of proteinsfused to the C-terminus of the peptide. Additionally, these datademonstrate that the R. reniformis luciferase protein is a highlyeffective reporter molecule, which is stable in a mammalian cellenvironment, and forms the basis of a sensitive, facile assay for geneexpression.

[0478] b. Expression of R. reniformis Luciferase Using pLNCX-ILRUCλ

[0479] To express the IL-2 signal peptide-R. reniformis fusion gene froman artificial mammalian chromosome, vector pLNCX-ILRUCλ is targeted forsite-specific integration into an artificial mammalian chromosomethrough homologous recombination of the lambda DNA sequences containedin the chromosome and the vector. This is accomplished by introductionof pLNCX-ILRUCλ into either a fusion cell line harboring mammalianartificial chromosomes or mammalian host cells that contain artificialmammalian chromosomes. If the vector is introduced into a fusion cellline harboring the artificial chromosomes, for example throughmicroinjection of the vector or transfection of the fusion cell linewith the vector, the cells are then grown under selective conditions,i.e. artificial chromosomes, which have incorporated vectorpLNCX-ILRUCλ, are isolated from the surviving cells, using purificationprocedures as described above, and then injected into the mammalian hostcells.

[0480] Alternatively, the mammalian host cells may first be injectedwith mammalian artificial chromosomes which have been isolated from afusion cell line. The host cells are then transfected with vectorpLNCX-ILRUCλ and grown.

[0481] The recombinant host cells are then assayed for luciferaseexpression as described above.

[0482] D. Other Targeting Vectors

[0483] These vector rely on positive and negative selection to insureinsertion and selection for the double recombinants. A single crossoverresults in incorporation of the DT-A, which kills the cell, doublecrossover recombinations delete the DT-1 gene.

[0484] 1. Plasmid pNEM1 contains:

[0485] DT-A: Diphtheria toxin gene (negative selectable marker)

[0486] Hyg: Hygromycin gene (positive selectable marker)

[0487] ruc: Renilla luciferase gene (non-selectable marker)

[0488] 1: LTR-MMTV promoter

[0489] 2: TK promoter

[0490] 3: CMV promoter

[0491] MMR: Homology region (plasmid pAG60)

[0492] 2. plasmid pNEM-2 and -3 are similar to pNEM 1 except fordifferent negative selectable markers:

[0493] pNEM-1: diphtheria toxin gene as “-” selectable marker

[0494] pNEM-2: hygromycin antisense gene as “-” selectable marker

[0495] pNEM-3: thymidine kinase HSV-1 gene as “-” selectable marker

[0496] 3. Plasmid—lambda DNA based homology:

[0497] pNEMA-1: base vector

[0498] pNEMA-2: base vector containing p5=gene

[0499] 1: LTR MMTV promoter

[0500] 2: SV40 promoter

[0501] 3: CMV promoter

[0502] 4: μTIIA promoter (metallothionein gene promoter)

[0503] - homology region (plasmid pAG60)

[0504] λ L.A. and λ R.A. homology regions for A left and right arms (λgt-WES).

EXAMPLE 13

[0505] Microinjection of Mammalian Cells with Plasmid DNA

[0506] These procedure will be used to microinject MACS into eukaryoticcells, including mammalian and insect cells.

[0507] The microinjection technique is based on the use of small glasscapillaries as a delivery system into cells and has been used forintroduction of DNA fragments into nuclei (see, e.g., Chalfie et al.(1994) Science 263:802-804). It allows the transfer of almost any typeof molecules, e.g., hormones, proteins, DNA and RNA, into either thecytoplasm or nuclei of recipient cells This technique has no cell typerestriction and is more efficient than other methods, includingCa²⁺-mediated gene transfer and liposome-mediated gene transfer. About20-30% of the injected cells become successfully transformed.

[0508] Microinjection is performed under a phase-contrast microscope. Aglass microcapillary, prefilled with the DNA sample, is directed into acell to be injected with the aid of a micromanipulator. An appropriatesample volume (1-10 pl) is transferred into the cell by gentle airpressure exerted by a transjector connected to the capillary. Recipientcells are grown on glass slides imprinted with numbered squares forconvenient localization of the injected cells.

[0509] a. Materials and Equipment

[0510] Nunclon tissue culture dishes 35×10 mm, Mouse cell line EC3/7C5Plasmid DNA pCH110 (Pharmacia), Purified Green Florescent Protein (GFP)(GFPs from Aequorea and Renilla have been purified and also DNA encodingGFPs has been cloned; see, e.g., Prasher et al. (1992) Gene 111:229-233;International PCT Application No. WO 95/07463, which is based on U.S.application Ser. No. 08/119,678 and U.S. application Ser. No.08/192,274), ZEISS Axiovert 100 microscope, Eppendorf transjector 5246,Eppendorf micromanipulator 5171, Eppendorf Cellocate coverslips,Eppendorf microloaders, Eppendorf femtotips and other standard equipment

[0511] b. Protocol

[0512] (1) Fibroblast cells are grown in Ø 35 mm tissue culture dishes(37° C., 5% CO₂) until the cell density reaches 80% confluency. Thedishes are removed from the incubator and medium to added to about a 5mm depth.

[0513] (2) The dish is placed onto the dish holder and the cellsobserved with 10× objective; the focus is desirably above the cellsurface.

[0514] (3) Plasmid or chromosomal DNA solution (1 ng/μl) and GFP proteinsolution are further purified by centrifuging the DNA sample at forcesufficient to removed any particular debris (typically about 10,000 rpmfor 10 minutes in a microcentrifuge).

[0515] (4) Two 2 μl of the DNA solution (1 ng/μl) is loaded into amicrocapillary with an Eppendorf microloader. During loading, the loaderis inserted to the tip end of the microcapillary. GFP (1 mg/ml) isloaded wit the same procedure.

[0516] (5) The protecting sheath is removed from the microcapillary andthe microcapillary is fixed onto the capillary holer connected with themicromanipulator.

[0517] (6) The capillary tip lowered to the surface of the medium andfocus on the cells gradually until the tip of the capillary reaches thesurface of a cell. Lower the capillary further so that the capillary isinserted into the cell. Various parameters, such as the level of thecapillary, the time and pressure are determined for the particularequipment. For example, using the fibroblast cell line C5 and theabove-noted equipment, the best conditions are: injection time 0.4second, pressure 80 psi. DNA can then be automatically injected into thenuclei of the cells.

[0518] (7) After injection, the cells are returned to the incubator, andincubated for about 18-24 hours.

[0519] (8) After incubation the number of transformants can bedetermined by a suitable method, which depends upon the selectionmarker. For example, if green fluorescent protein is used, the assay canbe performed using UV light source and fluorescent filter set at 0-24hours after injection. If β-gal-containing DNA, such as DNA-derived frompHC110, has been injected, then the transformants can be assayed forβ-gal.

[0520] i. Detection of β-galactosidase in cells injected with plasmidDNA. The medium is removed from the culture plate and the cells arefixed by addition of 5 ml of fixation Solution I: (1% glutaraldehyde;0.1 M sodium phosphate buffer, pH 7.0; 1 mM MgCl₂), and incubated for 15minutes at 37° C. Fixation Solution I is replaced with 5 ml of X-galSolution II: (0.2% X-gal, 10 mM sodium phosphate buffer (pH 7.0), 150 mMNaCl, 1 mM MgCl₂, 3.3 mM K₄Fe(CN)₆H₂O, 3.3 mM K₃Fe(CN)₆), and the platesare incubated for 30-60 minutes at 37° C.

[0521] The X-gal solution is removed and 2 ml of 70% glycerol is addedto each dish. Blue stained cells are identified under a lightmicroscope.

[0522] This will be used to introduce a MAC, particular the MAC with theanti-HIV megachromosome, to produce a mouse model for anti-HIV activity.

EXAMPLE 14

[0523] Transgenic Animals

[0524] Transgenic animals can be generated that express heterologousgenes which confer desired traits, e.g., disease resistance, in theanimals. A transgenic mouse is prepared to serve as model of adisease-resistant animal. Genes that encode vaccines or that encodetherapeutic molecules can be introduced into embryos or ES cells toproduce animals that express the gene product and thereby are resistantto or less susceptible to a particular disorder.

[0525] The mammalian artificial megachromosome can be used to generatetransgenic animals that stably express genes conferring desired traits,such as genes conferring resistance to pathogenic viruses. Transgenicmice containing a transgene encoding an anti-HIV ribozyme provide auseful model for the development of stable transgenic animals usingthese methods.

[0526] 1. Development of Control Transgenic Mice Expressing Anti-HIVRibozyme

[0527] Control transgenic mice are generated in order to comparestability and amounts of transgene expression in mice developed usingtransgene DNA carried on a vector (control mice) with expression in micedeveloped using transgenes carried in an artificial megachromosome.

[0528] a. Development of Control Transgenic Mice Expressingβ-Galactosidase

[0529] One set of control transgenic mice was generated bymicroinjection of mouse embryos with the β-galactosidase gene alone. Themicroinjection procedure used to introduce the plasmid DNA into themouse embryos is as described in Example 13, but modified for use withembryos (see, e.g., Hogan et al. (1994) Manipulating the Mouse Embryo: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., see, especially pages 255-264 and Appendix 3). Fertilizedmouse embryos (Strain CB6 obtained from Charles River Co.) were injectedwith 1 ng of plasmid pCH110 (Pharmacia) which had been linearized bydigestion with BamHI. This plasmid contains the β-galactosidase genelinked to the SV40 late promoter. The β-galactosidase gene productprovides a readily detectable marker for successful transgeneexpression. Furthermore, these control mice provide confirmation of themicroinjection procedure used to introduce the plasmid into the embryos.Additionally, because the megachromosome that is transferred to themouse embryos in the model system (see below) also contains theβ-galactosidase gene, the control transgenic mice that have beengenerated by injection of pCH110 into embryos serve as an analogoussystem for comparison of heterologous gene expression from a plasmidversus from a gene carried on an artificial chromosome.

[0530] After injection, the embryos are cultured in modified HTF mediumunder 5% CO₂ at 37° C. for one day until they divide to form two cells.The two-cell embryos are then implanted into surrogate mother femalemice (for procedures see, Manipulating the Mouse Embryo: A LaboratoryManual (1994) Hogan et al., eds., Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., pp. 127 et seq.).

[0531] b. Development of Control Transgenic Mice Expressing Anti-HIVRibozyme

[0532] One set of anti-HIV ribozyme gene-containing control transgenicmice was generated by microinjection of mouse embryos with plasmidpCEPUR-132 which contains three different genes: (1) DNA encoding ananti-HIV ribozyme, (2) the puromycin-resistance gene and (3) thehygromycin-resistance gene. Plasmid pCEPUR-132 was constructed byligating portions of plasmid pCEP-132 containing the anti-HIV ribozymegene (referred to as ribozyme D by Chang et al. ((1990) Clin. Biotech.2:23-31); see also U.S. Pat. No. 5,144,019 to Rossi et al., particularlyFIG. 4 of the patent) and the hygromycin-resistance gene with a portionof plasmid pCEPUR containing the puromycin-resistance gene.

[0533] Plasmid pCEP-132 was constructed as follows. Vector pCEP4(Invitrogen, San Diego, Calif.; see also Yates et al. (1985) Nature313:812-815) was digested with XhoI which cleaves in the multiplecloning site region of the vector. This ˜10.4-kb vector contains thehygromycin-resistance gene linked to the thymidine kinase gene promoterand polyadenylation signal, as well as the ampicillin-resistance geneand ColE1 origin of replication and EBNA-1 (Epstein-Barr virus nuclearantigen) genes and OriP. The multiple cloning site is flanked by thecytomegalovirus promoter and SV40 polyadenylation signal.

[0534] XhoI-digested pCEP4 was ligated with a fragment obtained bydigestion of plasmid 132 (see Example 4 for a description of thisplasmid) with XhoI and SalI. This XhoI/SalI fragment contains theanti-HIV ribozyme gene linked at the 3′ end to the SV40 polyadenylationsignal. The plasmid resulting from this ligation was designatedpCEP-132. Thus, in effect, pCEP-132 comprises pCEP4 with the anti-HIVribozyme gene and SV40 polyadenylation signal inserted in the multiplecloning site for CMV promoter-driven expression of the anti-HIV ribozymegene.

[0535] To generate pCEPUR-132, pCEP-132 was ligated with a fragment ofpCEPUR. pCEPUR was prepared by ligating a 7.7-kb fragment generated uponNheI/NruI digestion of pCEP4 with a 1.1-kb NheI/SnaBI fragment of pBabe(see Morgenstern and Land (1990) Nucleic Acids Res. 18:3587-3596 for adescription of pBabe) that contains the puromycin-resistance gene linkedat the 5′ end to the SV40 promoter. Thus, pCEPUR is made up of theampicillin-resistance and EBNA1 genes, as well as the ColE1 and OriPelements from pCEP4 and the puromycin-resistance gene from pBabe. Thepuromycin-resistance gene in pCEPUR is flanked by the SV40 promoter(from pBabe) at the 5′ end and the SV40 polyadenylation signal (frompCEP4) at the 3′ end.

[0536] Plasmid pCEPUR was digested with XhoI and SalI and the fragmentcontaining the puromycin-resistance gene linked at the 5′ end to theSV40 promoter was ligated with XhoI-digested pCEP-132 to yield the˜12.1-kb plasmid designated pCEPUR-132. Thus, pCEPUR-132, in effect,comprises pCEP-132 with puromycin-resistance gene and SV40 promoterinserted at the XhoI site. The main elements of pCEPUR-132 are thehygromycin-resistance gene linked to the thymidine kinase promoter andpolyadenylation signal, the anti-HIV ribozyme gene linked to the CMVpromoter and SV40 polyadenylation signal, and the puromycin-resistancegene linked to the SV40 promoter and polyadenylation signal. The plasmidalso contains the ampicillin-resistance and EBNA1 genes and the ColE1origin of replication and OriP.

[0537] Zygotes were prepared from (C57BL/6JxCBA/J) F1 female mice (see,e.g., Manipulating the Mouse Embryo: A Laboratory Manual (1994) Hogan etal., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., p. 429), which had been previously mated with a (C57BL/6JxCBA/J)F1 male. The male pronuclei of these F2 zygotes were injected (see,Manipulating the Mouse Embryo: A Laboratory Manual (1994) Hogan et al.,eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.)with pCEPUR-132 (˜3 μg/ml), which had been linearized by digestion withNruI. The injected eggs were then implanted in surrogate mother femalemice for development into transgenic offspring.

[0538] These primary carrier offspring were analyzed (as describedbelow) for the presence of the transgene in DNA isolated from tailcells. Seven carrier mice that contained transgenes in their tail cells(but that may not carry the transgene in all their cells, i.e., they maybe chimeric) were allowed to mate to produce non-chimeric or germ-lineheterozygotes. The heterozygotes were, in turn, crossed to generatehomozygote transgenic offspring.

[0539] 2. Development of Model Transgenic Mice Using MammalianArtificial Chromosomes

[0540] Fertilized mouse embryos are microinjected (as described above)with megachromosomes (1-10 pL containing 0-1 chromosomes/pL) isolatedfrom fusion cell line G3D5′ or H1D3′ (described above). Themegachromosomes are isolated as described herein. Megachromosomesisolated from either cell line carry the anti-HIV ribozyme (ribozyme D)gene as well as the hygromycin-resistance and β-galactosidase genes. Theinjected embryos are then developed into transgenic mice as describedabove.

[0541] Alternatively, the megachromosome-containing cell line G3D5* orH1D3* is fused with mouse embryonic stem cells (see, e.g., U.S. Pat. No.5,453,357, commercially available; see Manipulating the Mouse Embryo: ALaboratory Manual (1994) Hogan et al., eds., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., pages 253-289) followingstandard procedures see also, e.g., “Guide to Techniques in MouseDevelopment” in Methods in Enzymology Vol. 25, Wassarman and DePamphilis, eds. (1993), pages 803-932). (It is also possible to deliverisolated megachromosomes into embryonic stem cells using the Microcellprocedure (such as that described above).) The stem cells are culturedin the presence of a fibroblast (e.g., STO fibroblasts that areresistant to hygromycin and puromycin). Cells of the resultant fusioncell line, which contains megachromosomes carrying the transgenes (i.e.,anti-HIV ribozyme, hygromycin-resistance and β-galactosidase genes), arethen transplanted into mouse blastocysts, which are in turn implantedinto a surrogate mother female mouse where development into a transgenicmouse will occur.

[0542] Mice generated by this method are chimeric in that the transgeneswill be expressed in only certain areas of the mouse, e.g., the head,and thus may not be expressed in all cells.

[0543] 3. Analysis of Transgenic Mice for Transgene Expression

[0544] Beginning when the transgenic mice, generated as described above,are three-to-four weeks old, they can be analyzed for stable expressionof the transgenes that were transferred into the embryos (or fertilizedeggs) from which they develop. The transgenic mice may be analyzed inseveral ways as follows.

[0545] a. Analysis of Cells Obtained from the Transgenic Mice

[0546] Cell samples (e.g., spleen cells, lymphocytes, tail cells) areobtained from the transgenic mice. Any cells may be tested for transgeneexpression. If, however, the mice are chimeras generated bymicroinjection of fertilized eggs with fusions of embryonic stem cellswith megachromosome-containing cells, only cells from areas of the mousethat carry the transgene are expected to express the transgene. If thecells survive growth on hygromycin (or hygromycin and puromycin orneomycin, if the cells are obtained from mice generated by transfer ofboth antibiotic-resistance genes), this is one indication that they arestably expressing the transgenes. RNA isolated from the cells accordingto standard methods may also be analyzed by northern blot procedures todetermine if the cells express transcripts that hybridize to nucleicacid probes based on the antibiotic-resistance genes.

[0547] Additionally, cells obtained from the transgenic mice may also beanalyzed for β-galactosidase expression using standard assays for thismarker enzyme (for example, by direct staining of the product of areaction involving β-galactosidase and the X-gal substrate, see, e.g.,Jones (1986) EMBO J. 5:3133-3142, or by measurement of β-galactosidaseactivity, see, e.g., Miller (1972) in Experiments in Molecular Geneticspp. 352-355, Cold Spring Harbor Press). Analysis of β-galactosidaseexpression is particularly used to evaluate transgene expression incells obtained from control transgenic mice in which the only transgenetransferred into the embryo was the β-galactosidase gene.

[0548] Stable expression of the anti-HIV ribozyme gene in cells obtainedfrom the transgenic mice may be evaluated in several ways. First, DNAisolated from the cells according to standard procedures may besubjected to nucleic acid amplification using primers corresponding tothe ribozyme gene sequence. If the gene is contained within the cells,an amplified product of pre-determined size is detected uponhybridization of the reaction mixture to a nucleic acid probe based onthe ribozyme gene sequence. Furthermore, DNA isolated from the cells maybe analyzed using Southern blot methods for hybridization to such anucleic acid probe. Second, RNA isolated from the cells may be subjectedto northern blot hybridization to determine if the cells express RNAthat hybridizes to nucleic acid probes based on the ribozyme gene.Third, the cells may be analyzed for the presence of anti-HIV ribozymeactivity as described, for example, in Chang et al. (1990) Clin.Biotech. 2:23-31. In this analysis, RNA isolated from the cells is mixedwith radioactively labeled HIV gag target RNA which can be obtained byin vitro transcription of gag gene template under reaction conditionsfavorable to in vitro cleavage of the gag target, such as thosedescribed in Chang et al. (1990) Clin. Biotech. 2:23-31. After thereaction has been stopped, the mixture is analyzed by gelelectrophoresis to determine if cleavage products smaller in size thanthe whole template are detected; presence of such cleavage fragments isindicative of the presence of stably expressed ribozyme.

[0549] b. Analysis of Whole Transgenic Mice

[0550] Whole transgenic mice that have been generated by transfer of theanti-HIV ribozyme gene (as well as selection and marker genes) intoembryos or fertilized eggs can additionally be analyzed for transgeneexpression by challenging the mice with infection with HIV. It ispossible for mice to be infected with HIV upon intraperitoneal injectionwith high-producing HIV-infected U937 cells (see, e.g., Locardi et al.(1992) J. Virol. 66:1649-1654). Successful infection may be confirmed byanalysis of DNA isolated from cells, such as peripheral bloodmononuclear cells, obtained from transgenic mice that have been injectedwith HIV-infected human cells. The DNA of infected transgenic mice cellswill contain HIV-specific gag and env sequences, as demonstrated by, forexample, nucleic acid amplification using HIV-specific primers. If thecells also stably express anti-HIV ribozyme, then analysis of RNAextracts of the cells should reveal the smaller gag fragments arising bycleavage of the gag transcript by the ribozyme.

[0551] Additionally, the transgenic mice carrying the anti-HIV ribozymegene can be crossed with transgenic mice expressing human CD4 (i.e., thecellular receptor for HIV) (see Gillespie et al. (1993) Mol. Cell. Biol.13:2952-2958; Hanna et al. (1994) Mol. Cell. Biol. 14:1084-1094; andYeung et al. (1994) J. Exp. Med. 180:1911-1920, for a description oftransgenic mice expressing human CD4). The offspring of these crossedtransgenic mice expressing both the CD4 and anti-HIV ribozyme transgenesshould be more resistant to infection (as a result of a reduction in thelevels of active HIV in the cells) than mice expressing CD4 alone(without expressing anti-HIV ribozyme).

[0552] Since modifications will be apparent to those of skill in thisart, it is intended that this invention be limited only by the scope ofthe appended claims.

0 SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES:12 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 1293 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (iii) HYPOTHETICAL:NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: <Unknown> (vi) ORIGINALSOURCE: (ix) FEATURE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:GAATTCATCA TTTTTCANGT CCTCAAGTGG ATGTTTCTCA TTTNCCATGA TTTTAAGTT 60TCTCGCCATA TTCCTGGTCC TACAGTGTGC ATTTCTCCAT TTTNCACGTT TTNCAGTG 120TTCGTCATTT TCAAGTCCTC AAGTGGATGT TTCTCATTTN CCATGAATTT CAGTTTTC 180GCCATATTCC ACGTCCTACA GNGGACATTT CTAAATTTNC CACCTTTTTC AGTTTTCC 240GCCATATTTC ACGTCCTAAA ATGTGTATTT CTCGTTTNCC GTGATTTTCA GTTTTCTC 300CAGATTCCAG GTCCTATAAT GTGCATTTCT CATTTNNCAC GTTTTTCAGT GATTTCGT 360TTTTTTCAAG TCGGCAAGTG GATGTTTCTC ATTTNCCATG ATTTNCAGTT TTCTTGNA 420ATTCCATGTC CTACAATGAT CATTTTTAAT TTTCCACCTT TTCATTTTTC CACGCCAT 480TTCATGTCCT AAAGTGTATA TTTCTCCTTT TCCGCGATTT TCAGTTTTCT CGCCATAT 540CAGGTCCTAC AGTGTGCATT CCTCATTTTT CACCTTTTTC ACTGATTTCG TCATTTTT 600AGTCGTCAAC TGGATCTTTC TAATTTTCCA TGATTTTCAG TTATCTTGTC ATATTCCA 660TCCTACAGTG GACATTTCTA AATTTTCCAA CTTTTTCAAT TTTTCTCGAC ATATTTGA 720TGCTAAAGTG TGTATTTCTT ATTTTCCGTG ATTTTCAGTT TTCTCGCCAT ATTCCAGG 780CTAATAGTGT GCATTTCTCA TTTTTCACGT TTTTCAGTGA TTTCGTCATT TTTTCCAG 840GTCAAGGGGA TGTTTCTCAT TTTCCATGAG TGTCAGTTTT CTTGCTATAT TCCATGTC 900ACAGTGACAT TTCTAAATAT TATACCTTTT TCAGTTTTTC TCACCATATT TCACGTCC 960AAGTATATAT TTCTCATTTT CCCTGATTTT CAGTTTCCTT GCCATATTCC AGGTCCT 1020GTGTGCATTT CTCATTTTTC ACGTTTTTCA GTAATTTCTT CATTTTTTAA GCCCTCA 1080GGATGTTTCT CATTTTCCAT GATTTTCAGT TTTCTTGCCA TATACCATGT CCTACAG 1140ACATTTCTAA ATTATCCACC TTTTTCAGTT TTTCATCGGC ACATTTCACG TCCTAAA 1200TGTATTTCTA ATTTTCAGTG ATTTTCAGTT TTCTCGCCAT ATTCCAGGAC CTACAGT 1260CATTTCTCAT TTTTCACGTT TTTCAGTGAA TTC 1293 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1044 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: Genomic DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v)FRAGMENT TYPE: <Unknown> (vi) ORIGINAL SOURCE: (ix) FEATURE: (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 2: AGGCCTATGG TGAAAAAGGA AATATCTTCCCCTGAAAACT AGACAGAAGG ATTCTCAGA 60 TCTTATTTGT GATGTGCGCC CCTCAACTAACAGTGTTGAA GCTTTCTTTT GATAGAGC 120 TTTTGAAACA CTCTTTTTGT AAAATCTGCAAGAGGATATT TGGATAGCTT TGAGGATT 180 CGTTGGAAAC GGGATTGTCT TCATATAAACCCTAGACAGA AGCATTCTCA GAAGCTTC 240 TGGGATGTTT CAGTTGAAGT CACAGTGTTGAACAGTCCCC TTTCATAGAG CAGGTTTG 300 ACACTCTTTT TTGTAGTATC TGGAAGTGGACATTTGGAGC GATCTCAGGA CTGCGGTG 360 AAAGGAAATA TCTTCCAATA AAAGCTAGATAGAGGCAATG TCAGAAACCT TTTTCATG 420 GTATCTACTC AGCTAACAGA GTTGAACCTTCCTTTGAGAG AGCAGTTTTG AAACACTC 480 TTTGTGGAAT CTGCAAGTGG ATATTTGTCTAGCTTTGAGG ATTTCGTTGG GAAACGGG 540 TACATATAAA AAGCAGACAG CAGCATTCCCAGAAACTTCT TTGTGATGTT TGCATTCA 600 TCACAGAGTT GAACATTCCC TTTCATAGAGCAGGTTTGAA ACACACTTTT TGATGTAT 660 GGATGTGGAC ATTTGCAGCG CTTTCAGGCCTAAGGTGAAA AGGAAATATC TTCCCCTG 720 AACTAGACAG AAGCATTCTC AGAAACTTATTTGTGATGTG CGCCCTCAAC TAACAGTG 780 GAAGCTTTCT TTTGATAGAG GCAGTTTTGAAACACTCTTT TGTGGAATCT GCAAGTGG 840 ATTTGTCTAG CTTTGAGGAT TTCTTTGGAAACGGGATTAC ATATAAAAAG CAGACAGC 900 CATTCCCAGA ATCTTGTTTG TGATGTTTGCATTCAAGTCA CAGAGTTGAA CATTCCCT 960 CAGAGAGCAG GTTTGAACAC TCTTTTTATAGTATCTGGAT GTGGACATTT GGAGCGC 1020 CAGGGGGGAT CCTCTAGAAT TCCT 1044 (2)INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:2492 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: <Unknown> (vi) ORIGINAL SOURCE:(ix) FEATURE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: CTGCAGCTGGGGGTCTCCAA TCAGGCAGGG GCCCCTTACT ACTCAGATGG GGTGGCCGA 60 TAGGGGAAGGGGGTGCAGGC TGCATGAGTG GACACAGCTG TAGGACTACC TGGGGGCT 120 GGATCTATGGGGGTGGGGAG AAGCCCAGTG ACAGTGCCTA GAAGAGACAA GGTGGCCT 180 GAGGGTCTGAGGAACATAGA GCTGGCCATG TTGGGGCCAG GTCTCAAGCA GGAAGTGA 240 AATGGGACAGGCTTGAGGAT ACTCTACTCA GTAGCCAGGA TAGCAAGGAG GGCTTGGG 300 TGCTATCCTGGGGTTCAACC CCCCAGGTTG AAGGCCCTGG GGGAGATGGT CCCAGGAC 360 ATTACAATGGACACAGGAGG TTGGGACACC TGGAGTCACC AAACAAAACC ATGCCAAG 420 AGACCATGAGTAGGGGTGTC CAGTCCAGCC CTCTGACTGA GCTGCATTGT TCAAATCC 480 AGGGCCCCTGCTGCCACCTA GTGGCTGATG GCATCCACAT GACCCTGGGC CACACGCG 540 TAGGGTCTCTGTGAAGACCA AGATCCTTGT TACATTGAAC GACTCCTAAA TGAGCAGA 600 TTTCCACCTATTCGAAACAA TCACATAAAA TCCATCCTGG AAAAAGCCTG GGGGATGG 660 CTAAGGCTAGGGATAGGGTG GGATGAAGAT TATAGTTACA GTAAGGGGTT TAGGGTTA 720 GATCAACGTTGGTTAGGAGT TAGGGATACA GTAGGGTACC GGTAGGGTTA GGGGTTAG 780 TTAGGGGTTAGGGTTAGGGT TAGGGTTAGG GTTAGGGTTA GGGGTTAGGG GTTAGGGT 840 GGGTTAGGTTTTGGGGTGGC GTATTTTGGT CTTATACGCT GTGTTCCACT GGCAATGA 900 AGAGTTCTTGTTTTTCCTTC AGCAATTTGT CATTTTTAAA AGAGTTTAGC AATTCTAA 960 GATATAGACCAGCTGTGCTA TCTCATTGTG GTTTTCAATT GTAACCACAT TGTGGTT 1020 ATGTGTTTACTTGCCATCTG TAGATCTTCT TTGCGTGAGG TGTCTGTTCA GATGTGT 1080 CATTTCTTGNNTTTNGGCTG TTTAACTTAT TGTTTAGTTT TAATAATTTT TTATATA 1140 GAAGACAAATCTTTCTCAGA TGTGTATTTG CAAATATTTC TTCAATATGA GGCTTGC 1200 TGTCTCTAACAAGGTCTCTT CAGAGATAAC TTAAATATAA GAAATCCACA CTGTCAC 1260 TTTTGTGTATATCTACCTTT TGTGTCATTT GTTAAAATTC ATTACCAAAC CCAAAGG 1320 ATAGCTTTTCTTCTATTGTT TCTTCTAGAA ATTTGTATAG TTTTGCATTT TTAGTGT 1380 GATGATTTTGAGTGATTATT TGTGTAAGTT GTAAAGTTTT CGTCTATATC CATATCA 1440 CTTATGGTTTCCAATTAATC GTTCCCTCAC TATTTTTGGG AAAGACACAG GATAGTG 1500 TTTGTTAGAGTAGATAGGTA GCTAGACATG AACAGGAGGG GGCCTCCTGG AAAAGGG 1560 GTCTGGGAAGGCTCACCTGG AGGACCACCA AAAATTCACA TATTAGTAGC ATCTCTA 1620 CTGGAGTGGATGGGCACTTG TCAATTGTGG GTAGGAGGGA AAAGAGGTCC TATGCAG 1680 GAAACTCCCTAGAACTCCTC TGAAGATGCC CCAATCATTC ACTCTGCAAT AAAAATG 1740 GAATATTGCTAGCTACATGC TGATAAGGNN AAAGGGGACA TTCTTAAGTG AAACCTG 1800 CCATAAGTACAGATTAGGGC AGAGAAGGAC ATTCAAAAGA GGCAGGCGCA GTAGGTA 1860 ACGTGATCGCTGTCAGTGTG CCTGGGATGG CGGGAAGGAG GCTGGTGCCA GAGTGGA 1920 GTATTGATCACCACACATAT ACCTCAACCA ACAGTGAGGA GGTCCCACAA GCCTAAG 1980 GGCAAGTTGGGGAGCTAAGG CAGTAGCAGG AAAACCAGAC AAAGAAAACA GGTGGAG 2040 TGAGACAGAGGCAGGAATGT GAAGAAATCC AAAATAAAAT TCCCTGCACA GGACTCT 2100 GCTGTTTAATGCATCGCTCA GTCCCACTCC TCCCTATTTT TCTACAATAA ACTCTTT 2160 CTGTGTTTCTTTTCAATGAA GTTATCTGCC ATCTTTGTAT TGCCTCTTGG TGAAAAT 2220 TCTTCCAAGTTAAACAAGAA CTGGGACATC AGCTCTCCCC AGTAATAGCT CCGTTTC 2280 TTGAATTTACAGAACTGATG GGCTTAATAA CTGGCGCTCT GACTTTAGTG GTGCAGG 2340 CCGTCACACCGGGACCAAGA GTGCCCTGCC TAGTCCCCAT CTGCCCGCAG GTGGCGG 2400 CCTCGACACTGACAGCAATA GGGTCCGGCA GTGTCCCCAG CTGCCAGCAG GGGGCGT 2460 ACGACTACACTGTGAGCAAG AGGGCCCTGC AG 2492 (2) INFORMATION FOR SEQ ID NO: 4: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:Genomic DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENTTYPE: <Unknown> (vi) ORIGINAL SOURCE: (ix) FEATURE: (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 4: GGGGAATTCA TTGGGATGTT TCAGTTGA 28 (2)INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: <Unknown> (vi) ORIGINAL SOURCE:(ix) FEATURE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: CGAAAGTCCCCCCTAGGAGA TCTTAAGGA 29 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 47 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: <Unknown> (vi)ORIGINAL SOURCE: (ix) FEATURE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:CGCTTAATA CTCTGATGAG TCCGTGAGGA CGAAACGCTC TCGCACC 47 (2) INFORMATIONFOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: Genomic DNA (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (v) FRAGMENT TYPE: <Unknown> (vi) ORIGINAL SOURCE: (ix)FEATURE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: CGATTTAAAT TAATTAAGCCCGGGC 25 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: <Unknown> (vi)ORIGINAL SOURCE: (ix) FEATURE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:AAATTTAAT TAATTCGGGC CCGTCGA 27 (2) INFORMATION FOR SEQ ID NO: 9: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 69 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: ATG TAC AGG ATG CAACTC CTG TCT TGC ATT GCA CTA AGT CTT GCA CTT 48 Met Tyr Arg Met Gln LeuLeu Ser Cys Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 GTC ACA AAC AGT GCACCT ACT 69 Val Thr Asn Ser Ala Pro Thr 20 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 945 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: cDNA (vi) ORIGINAL SOURCE: (ix) FEATURE: (A) NAME/KEY: CodingSequence (B) LOCATION: 1...942 (D) OTHER INFORMATION: Renilla ReinformisLuciferase (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: PATENT NO.:5,418,155 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: AGC TTA AAG ATG ACTTCG AAA GTT TAT GAT CCA GAA CAA AGG AAA CGG 48 Ser Leu Lys Met Thr SerLys Val Tyr Asp Pro Glu Gln Arg Lys Arg 1 5 10 15 ATG ATA ACT GGT CCGCAG TGG TGG GCC AGA TGT AAA CAA ATG AAT GTT 96 Met Ile Thr Gly Pro GlnTrp Trp Ala Arg Cys Lys Gln Met Asn Val 20 25 30 CTT GAT TCA TTT ATT AATTAT TAT GAT TCA GAA AAA CAT GCA GAA AAT 144 Leu Asp Ser Phe Ile Asn TyrTyr Asp Ser Glu Lys His Ala Glu Asn 35 40 45 GCT GTT ATT TTT TTA CAT GGTAAC GCG GCC TCT TCT TAT TTA TGG CGA 192 Ala Val Ile Phe Leu His Gly AsnAla Ala Ser Ser Tyr Leu Trp Arg 50 55 60 CAT GTT GTG CCA CAT ATT GAG CCAGTA GCG CGG TGT ATT ATA CCA GAT 240 His Val Val Pro His Ile Glu Pro ValAla Arg Cys Ile Ile Pro Asp 65 70 75 80 CTT ATT GGT ATG GGC AAA TCA GGCAAA TCT GGT AAT GGT TCT TAT AGG 288 Leu Ile Gly Met Gly Lys Ser Gly LysSer Gly Asn Gly Ser Tyr Arg 85 90 95 TTA CTT GAT CAT TAC AAA TAT CTT ACTGCA TGG TTG AAC TTC TTA ATT 336 Leu Leu Asp His Tyr Lys Tyr Leu Thr AlaTrp Leu Asn Phe Leu Ile 100 105 110 TAC CAA AGA AGA TCA TTT TTT GTC GGCCAT GAT TGG GGT GCT TGT TTG 384 Tyr Gln Arg Arg Ser Phe Phe Val Gly HisAsp Trp Gly Ala Cys Leu 115 120 125 GCA TTT CAT TAT AGC TAT GAG CAT CAAGAT AAG ATC AAA GCA ATA GTT 432 Ala Phe His Tyr Ser Tyr Glu His Gln AspLys Ile Lys Ala Ile Val 130 135 140 CAC GCT GAA AGT GTA GTA GAT GTG ATTGAA TCA TGG GAT GAA TGG CCT 480 His Ala Glu Ser Val Val Asp Val Ile GluSer Trp Asp Glu Trp Pro 145 150 155 160 GAT ATT GAA GAA GAT ATT GCG TTGATC AAA TCT GAA GAA GGA GAA AAA 528 Asp Ile Glu Glu Asp Ile Ala Leu IleLys Ser Glu Glu Gly Glu Lys 165 170 175 ATG GTT TTG GAG AAT AAC TTC TTCGTG GAA ACC ATG TTG CCA TCA AAA 576 Met Val Leu Glu Asn Asn Phe Phe ValGlu Thr Met Leu Pro Ser Lys 180 185 190 ATC ATG AGA AAG TTA GAA CCA GAAGAA TTT GCA GCA TAT CTT GAA CCA 624 Ile Met Arg Lys Leu Glu Pro Glu GluPhe Ala Ala Tyr Leu Glu Pro 195 200 205 TTC AAA GAG AAA GGT GAA GTT CGTCGT CCA ACA TTA TCA TGG CCT CGT 672 Phe Lys Glu Lys Gly Glu Val Arg ArgPro Thr Leu Ser Trp Pro Arg 210 215 220 GAA ATC CCG TTA GTA AAA GGT GGTAAA CCT GAC GTT GTA CAA ATT GTT 720 Glu Ile Pro Leu Val Lys Gly Gly LysPro Asp Val Val Gln Ile Val 225 230 235 240 AGG AAT TAT AAT GCT TAT CTACGT GCA AGT GAT GAT TTA CCA AAA ATG 768 Arg Asn Tyr Asn Ala Tyr Leu ArgAla Ser Asp Asp Leu Pro Lys Met 245 250 255 TTT ATT GAA TCG GAT CCA GGATTC TTT TCC AAT GCT ATT GTT GAA GGC 816 Phe Ile Glu Ser Asp Pro Gly PhePhe Ser Asn Ala Ile Val Glu Gly 260 265 270 GCC AAG AAG TTT CCT AAT ACTGAA TTT GTC AAA GTA AAA GGT CTT CAT 864 Ala Lys Lys Phe Pro Asn Thr GluPhe Val Lys Val Lys Gly Leu His 275 280 285 TTT TCG CAA GAA GAT GCA CCTGAT GAA ATG GGA AAA TAT ATC AAA TCG 912 Phe Ser Gln Glu Asp Ala Pro AspGlu Met Gly Lys Tyr Ile Lys Ser 290 295 300 TTC GTT GAG CGA GTT CTC AAAAAT GAA CAA TAA 945 Phe Val Glu Arg Val Leu Lys Asn Glu Gln 305 310 (2)INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: <Unknown> (vi) ORIGINAL SOURCE:(ix) FEATURE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: TTTGAATTC ATGTACAGGAT GCAACTCCTG 30 (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: GenomicDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE:<Unknown> (vi) ORIGINAL SOURCE: (ix) FEATURE: (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 12: TTTGAATTCA GTAGGTGCAC TGTTTGTCAC 30

What is claimed:
 1. A method for producing a transgenic non-humananimal, comprising: introducing a cell comprising a satellite artificialchromosome into a female non-human animal, wherein the cell developsinto an embryo in a female non-human animal; and allowing the embryo todevelop into a transgenic non-human animal comprising a satelliteartificial chromosome.
 2. The method of claim 1, wherein the satelliteartificial chromosome comprises heterologous DNA that encodes atherapeutic product.
 3. The method of claim 1, wherein the cell containsthe satellite artificial chromosome in a pronucleus.
 4. The method ofclaim 1, wherein the cell is a zygote.
 5. The method of claim 1, whereinthe cell is a fertilized ovum.
 6. The method of claim 1, wherein thecell is an ovum that develops into an embryo or zygote.
 7. The method ofclaim 1, wherein the cell is a bird, mouse, reptile, amphibian, insector fish cell.
 8. A method of producing a transgenic non-human animalembryo, comprising: introducing a satellite artificial chromosome into acell, wherein the cell develops in culture into a non-human animalembryo; and culturing the cell under conditions whereby it develops intoan embryo.
 9. The method of claim 8, wherein the cell comprises afertilized oocyte, an ovum, a fertilized ovum or a zygote.
 10. Themethod of claim 8, wherein the cell is a bird, mouse, reptile,amphibian, insect, or fish cell.
 11. The method of claim 1, wherein thesatellite artificial chromosome is isolated prior to introduction intothe cell. 12 The method of claim 1, wherein the satellite artificialchromosome is introduced into the cell by a method selected from thegroup consisting of direct uptake, incubation with polyethylene glycol(PEG), lipofection, microinjection, cell fusion, microcell fusion,electroporation, electrofusion, particle bombardment, projectilebombardment, calcium phosphate precipitation and site-specifictargeting.
 13. The method of claim 1, wherein the embryo and the femalenon-human animal are of the same species.
 14. A method for producing atransgenic non-human animal, comprising: introducing an embryocomprising a satellite artificial chromosome into a female non-humananimal; and allowing the embryo to develop into a transgenic non-humananimal comprising a satellite artificial chromosome.
 15. The method ofclaim 14, wherein: the embryo is produced by introducing a satelliteartificial chromosome into a cell that develops into an embryo; andintroduction is effected by a method selected from the group consistingof direct uptake, incubation with polyethylene glycol (PEG),lipofection, microinjection, cell fusion, microcell fusion,electroporation, electrofusion, particle bombardment, projectilebombardment, calcium phosphate precipitation and site-specifictargeting.
 16. The method of claim 15, wherein the cell is selected fromthe group consisting of a bird, insect or fish cell.
 17. The method ofclaim 15, wherein the embryo and the female non-human animal are of thesame species.
 18. A method for producing a transgenic non-human animal,comprising: introducing a fertilized oocyte comprising a satelliteartificial chromosome into a female non-human animal; and allowing theresulting embryo to develop into a transgenic non-human animalcomprising a satellite artificial chromosome.
 19. A method for producinga transgenic animal, comprising: introducing an embryonic stem cellcomprising a satellite artificial chromosome into an embryo; introducingthe embryo into a female mouse; and allowing the embryo to develop intoa transgenic animal comprising a satellite artificial chromosome. 20.The method of claim 19, wherein the animal is a bird.
 21. A method forproducing a transgenic non-human animal, comprising: introducing an ovumcomprising a satellite artificial chromosome (SATAC) into a femalenon-human animal, wherein the ovum develops into a zygote or embryo; andallowing the embryo or zygote to develop into a transgenic non-humananimal comprising the SATAC.
 22. The method of claim 21, wherein thesatellite artificial chromosome is a megachromosome derived from a cellline having all of the identifying characteristics of the cell linedeposited under ECACC accession number 96040928 or 96040929.